Formulation and process for modulating wound healing

ABSTRACT

Methods and compounds are disclosed for wound healing by modulating autophagy. A formulation for modulating autophagy comprises a first modulating compound (FAM) selected from compounds having the general structure (I): 
     
       
         
         
             
             
         
       
         
         
           
             wherein: L represents a linker selected from —C≡C—, (a tolan), —CH═CH— (a stilbene, preferably trans); or —CR a ═CR b — a stilbene derivative; where R a  and R b  are independently H or phenyl optionally substituted with —(R 3 ) p  or —(R 4 ) q ; 
           
         
       
    
     R 1  to R 4  are independent substituents at any available position of the phenyl rings, preferably at 3, 3′, 4, 4′, and/or 5, 5′; and m, n, p, and q are independently 0, 1, 2, or 3 representing the number of substituents of the rings, respectively, but at least one of m or n must be ≥1. Each R 1  to R 2  is independently selected from substituents described herein, including but not limited to hydroxyl, alkoxy, halo, halomethyl and glycosides. The formulation may also include an auxiliary autophagy modulating compound (AAM) as described herein. The formulation may include a hydrogel formed by the compounds themselves or otherwise and may include salts and/or complexes.

This application is a continuation application of U.S. application Ser.No. 15/093,146, filed under 35 U.S.C. § 111(a) on Apr. 7, 2016, now U.S.Pat. No. 10,045,950; which claims the priority to U.S. ProvisionalApplication No. 62/144,539, filed under 35 U.S.C. § 111(b) on Apr. 8,2015. The entire disclosures of all the aforementioned applications areexpressly incorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

The present invention relates generally to the field of cellular biologyand, more particularly, to compounds, formulations, and methods formodulating autophagy to treat a wound or skin related disease orcondition. The name, “autophagy,” also known as autophagocytosis, isderived from the Greek words meaning “eat” and “self.” Autophagy isgenerally defined as self-digestion.

Autophagy is primarily a lysosomal salvage or recycling pathway that iscommonly used by cells to perform homeostatic functions by degradingaging proteins and organelles and reabsorbing nucleotides, amino acids,and free fatty acids for new molecule synthesis and ATP production.Autophagy may be up-regulated in response to extra- or intracellularstress and signals such as starvation, growth factor deprivation, ERstress, and pathogen infection as a cellular-level self-preservationmechanism. In some pathologic conditions, it may be desirable tostimulate the autophagy process, while in other pathologic conditions,such as wound healing, it may be desirable to suppress or slow theautophagy process to reduce the destruction of cells. Thus, modulationof autophagy may invoke either enhancing/stimulating orsuppressing/slowing the cell growth and cell death process.

The most common form of autophagy involves (1) the formation of anisolation membrane, which extends and the termini ultimately (2) fuse toencompass the cellular contents within a double-membrane vesicle knownas an autophagosome. The autophagosome then (3) fuses with a lysosomethat provides enzymes to (4) digest the contents of the autophagosome,which contents then become available to the cell again as raw materialsor building block nutrients. Autophagy exhibits some similarities to theparallel proteasome degradation of ubiquitin-tagged proteins, butdiffers in that the autophagosome contains not only proteins, but alsocytoplasm, mitochondria, organelles and other cellular structures. Inthis sense it is known as a bulk degradation system.

Since the process of autophagy can be both beneficial and detrimental tothe cell depending on external factors and conditions, the process mustbe tightly regulated. Both yeast and mammalian systems have been studiedand utilize up to 36 proteins. FIG. 1 illustrates the process inmammals.

The yeast autophagy-related gene product (Atg8) has three mammalianhomologues: (1) LC3, (2) GABAA receptor-associated protein (GABARAP),and (3) Golgi-associated ATPase enhancer (GATE-16). Among them, LC3 ismost actively studied and frequently used as a mammalian autophagymarker. Shortly after translation (proLC3), LC3 is processed at theC-terminus by Atg4A or Atg4B into LC3-I. Upon induction or enhancementof autophagy, LC3-I is conjugated to the substratephosphatidylethanolamine (PE) via E1 (yeast Atg7) and E2 (yeast Atg3).The PE-LC3-I conjugate is referred to as LC3-II. This conjugation occursin the process at the point of autophagosome formation and leads to theconversion of soluble LC3-I to the autophagic vesicle associated LC3-II.This lipid conjugation allows LC3-II to be used as a marker of autophagyactivity.

Wound progression is caused by many mechanisms including local tissuehypo-perfusion, prolonged inflammation, free radical damage, apoptosis,and necrosis. These are typically broken up into three stages followingthe injury including; (i) the inflammation phase, (ii) cellproliferation phase and (iii) remodeling phase. Each one of these phasesis linked to a biologically and histologically unique fingerprint andautophagy plays a special role at each phase. For example, during theinflammation stage, autophagy is initially protective of tissue in thatit attempts to keep the wound edge cells from dying. In theproliferative stage, during which involves cell multiplication andmigration of multiple cell types to close the wound, autophagy helps toregulate β1-integrins and other cell migration proteins to form acontrolled line of cells at the leading edge of the wound. Inremodeling, the direction of these migrating cells is often controlledby the directionality and deposition of collagen that is secreted bythese transformed fibroblasts as pro-collagen. Collagen along with othercomponents such as fibronectin and lamin help regenerate the basementmembrane during the proliferation and migration stages.

During the wound healing process collagen fibrils provide the structuraltopology, rigidity and organization that allows for proper cellmigration. In chronic wounds and burns this environment is subjected toa variety of factors including an altered pH, chronic inflammation andoften a loss in the basement membrane including the essential collagentracts that are used by migrating wound cells (e.g. keratinocytes,fibroblasts, myofibroblasts) to fill the wound and restore a layer ofintact skin to protect the body from invading microbes and environmentalfactors (e.g. temperature regulation). During the natural healingprocess skin cells secrete the soluble form of collagen known aspro-collagen that can form higher order structures enzymatically orundergo an entropy driven self-assembly process (Prockop, D. and D.Hulmes (1994). Pro-collagen is secreted from the cell as a triple helix,which can self-assemble into fibrils due to its liquid crystal nature.This process is highly dependent on the pH, ionic strength, temperature,and concentration. Fibrils then are formed into individual collagenfibers, which are randomly assembled into higher order 3-dimensonalnetworks and structures.

U.S. Pat. Nos. 6,599,945 and 7,094,809 disclose several hydroxytolancompounds and their use in inhibiting the formation of infectious herpesvirus particles or for treating gonorrhea caused by Neisseriagonorrhoeae. WO2009/126700 discloses the use of similar compounds forskin care, such as UV radiation, and cosmetic uses. And U.S. Pat. No.8,716,355 (WO2011/0130468) and U.S. Pat. No. 8,680,142 (WO2011/0160301)disclose similar hydroxytolans for use as anti-tumor agents. However,the potential utility of these, or any other hydroxytolans asautophagy-modulating compounds was unknown until the making of thepresent invention. U.S. Pat. Nos. 6,008,260, 6,197,834 and 6,355,692disclose certain hydroxylated stilbenes, and specifically resveratrol.None of these references disclose the use of such modified tolan orstilbene compounds as autophagy modulating agents.

It would be advantageous if the process of autophagy could be modulated,that is stimulated or enhanced in some conditions, and slowed orsuppressed in other conditions.

SUMMARY OF THE INVENTION

The present invention relates to compounds, formulations, and methodsfor modulating autophagy, particularly for the indication or purpose ofpromoting wound healing in a patient having a chronic wound or skincondition. In one aspect, the invention comprises a method of promotingwound healing comprising administering at least one first autophagymodulating (FAM) compound as described herein. In most embodiments forpromotion of wound healing, the autophagy modulation is directionallyupregulation of autophagy activity. In certain embodiments, the FAM isadministered with an auxiliary autophagy modulator (AAM) compound. TheFAM and AAM may be co-administered or administered one prior to theother. If co-administered, they may be formulated in the same dosageform or as two distinct dosages or drug products. The AAM may modulatethe effect of the FAM by either stimulating or increasing its effect, orby depressing or inhibiting its effect since many are hormeticcompounds.

These autophagy modulators are liquid crystalline in nature and act inspecific combinations to treat a wound or skin related disease orcondition. The invention further describes the use of these liquidcrystals to create bioactive formulations such as hydrogels andalginates that can also serve as bioactive membranes to promote healingin a wound or skin related condition. Thus, in some aspects, theinvention comprises a formulation containing a FAM and AAM. Theformulation may be a hydrogel, such as an alginate. The formulation mayinclude a cationic salt of an FAM, or a complex of an FAM and an AAMwith a cation. In other formulations, the FAM and/or AAM may beformulated with a cyclodextrin.

The specific FAM may be a tolan or stilbene (including cis and transstilbenes) and may have any of a variety of substituents as describedherein. In some embodiments, the substituents are hydroxyl or alkoxylgroups that increase solubility and polarity of the molecule. Any FAMmay be administered with any AAM. For example, the FAM may be a tolanand the AAM may be a vitamin, acidic sugar, amino acid, or quinoloneLikewise, the FAM may be a stilbene and the AAM may be a vitamin, acidicsugar, amino acid, or quinolone.

In some aspects, the method promotes wound healing in specificindications, such as wherein the wound or skin condition is one or moreselected from: aging, autoimmune diseases with inflammation, avascularnecrosis, bacterial infection, cancers, diabetic neuropathies,endometriosis, fungal infection, gout, hairloss, infectious arthritis,inflammation, inflammatory bowel, ischemia, Lyme disease, organ/tissuetransplant, parasitic infection, psoriatic arthritis, psoriasis,pseudogout, rheumatoid arthritis, scleraderma, scurvy, sepsis, skindiseases, surgical scars, surgical adhesions, transfection procedures,ulcerative colitis, ulcers, viral infection, warts, surgical wounds,incisions, lacerations, cuts and scrapes donor site wounds from skintransplants, traumatic wounds, infectious wounds, ischemic wounds,burns, bullous wounds, aseptic wounds, contused wounds, incised wounds,lacerated wounds, non-penetrating wounds, open wounds, penetratingwounds, perforating wounds, puncture wounds, septic wounds, subcutaneouswounds, chronic ulcers, gastric ulcers, skin ulcers, peptic ulcer,duodenal ulcer, gastric ulcer, gouty ulcer, hypertensive ischemic ulcer,stasis ulcer, sublingual ulcer, submucous ulcer, symptomatic ulcer,trophic ulcer, tropical ulcer and veneral ulcer.

In some methods, the wound to be healed is dematological in nature, suchas cuts, abrasions, ulcers of many types and degrees, aging, skininelasticity, and the like. In other methods, the wound may beophthalmic or otic; in other methods, the wound may be oral in nature,such as cancer sores, herpes viral infections, tooth extraction wounds,gingivitis, or the like.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, incorporated herein and forming a part of thespecification, illustrate the present invention in its several aspectsand, together with the description, serve to explain the principles ofthe invention. In the drawings, the thickness of the lines, layers, andregions may be exaggerated for clarity.

FIG. 1 is a schematic illustration showing the role of LC3 in autophagy.

FIG. 2 is a schematic illustration showing autophagy related humandiseases.

FIG. 3 is a bar graph of LC3-II fluorescent staining as described inExample 5.

FIG. 4 is a bar graph of an LC3-II Western Blot as described in Example6.

FIG. 5 is a bar graph of the LC3-II/LC3-I ratio as described in Example6.

FIG. 6 is a graph of AKT signaling as described in Example 8.

FIG. 7 is a graph of Fibroblast Growth Factor as described in Example 9.

The accompanying drawings, incorporated herein and forming a part of thespecification, illustrate the present invention in its several aspectsand, together with the description, serve to explain the principles ofthe invention. In the drawings, the thickness of the lines, layers, andregions may be exaggerated for clarity.

DETAILED DESCRIPTION

Numerical ranges, measurements and parameters used to characterize theinvention—for example, angular degrees, quantities of ingredients,polymer molecular weights, reaction conditions (pH, temperatures, chargelevels, etc.), physical dimensions and so forth—are necessarilyapproximations; and, while reported as precisely as possible, theyinherently contain imprecision derived from their respectivemeasurements. Consequently, all numbers expressing ranges of magnitudesas used in the specification and claims are to be understood as beingmodified in all instances by the term “about.” All numerical ranges areunderstood to include all possible incremental sub-ranges within theouter boundaries of the range. Thus, a range of 30 to 90 unitsdiscloses, for example, 35 to 50 units, 45 to 85 units, and 40 to 80units, etc. Unless otherwise defined, percentages are weight/weight(wt/wt); although formulations are generally weight/volume (w/v), ingrams per 100 mL (which is equivalent to wt/wt with aqueous solutionshaving a density of 1.0), and area of wounds is expressed in cm² asarea/area (a/a).

All patents, published patent applications, and non-patent literaturereferences cited herein are incorporated herein by reference in theirentirety.

In some aspects, the invention comprises methods of modulating autophagycomprising the administration of a first autophagy modulator (FAM)compound and optionally an auxiliary autophagy modulator (AAM) compound.The FAM and AAM compounds are described in more detail in sectionsbelow. They may be given sequentially or concomitantly. If givensequentially, the order may be FAM first, then AMM, or AMM first, thenFAM. If given concomitantly, they may be given in separate, individualdrug products or a single drug product as a combination of ingredients.The compounds may be administered from once daily up to about 6 timesper day, depending on the formulation excipients. Administration routesinclude topical, transdermal, oral, nasal, ophthalmic, otic, IV, IM,subcutaneous, rectal, and vaginal.

The use of pharmaceutical excipients in the preparation of drug productsis generally well understood from pharmaceutical treatises such asRemington's Pharmaceutical Sciences, 18^(th) Edition (1990) and itssubsequent editions, like Remingtons: The Science and Practice ofPharmacy, 22n^(d) edition (2012). Topical formulations may be combinedwith solvents, emulsifiers, emollients, solvents, etc. into solutions,suspensions, creams, ointments and hydrogels, among others.

In certain embodiments, the invention involves a formulation containinga FAM compound and an AAM compound. The relative amounts of FAM to AAMin a formulation expressed as a molar ratio may range from about 500:1to about 1:500 (FAM:AAM), or, in certain embodiments, from about 200:1to 1:200. In liquid formulations, the FAM may comprise from about 0.01%to about 40% (w/v) of the formulation and the AMM may comprise fromabout 0.01% to about 99.9% (w/v) of the formulation. In certainembodiments, the FAM may comprise from about from about 0.1% to about40% (w/v) of the formulation and the AMM may comprise from about 0.1% toabout 60% (w/v) of the formulation. Optimally the formulationconcentration of an FAM is between 0.5-15% (w/v).

Chemical and Biological Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described herein. All references cited herein,including books, journal articles, published U.S. or foreign patentapplications, issued U.S. or foreign patents, and any other references,are each incorporated by reference in their entireties, including alldata, tables, figures, and text presented in the cited references.

The following terms used throughout this application have the meaningsascribed below.

A “first autophagy modulator” or “FAM compound” or “FAM” means acompound of formula I:

wherein L is a linker selected from: —C≡C— and —CR_(a)═CR_(b)—;

-   -   R_(a) and R_(b) are independently H or phenyl optionally        substituted with —(R³)_(p) or —(R⁴)_(q);    -   R¹ to R⁴ are independently substituents at any available        position of the phenyl rings;    -   m, n, p and q are, independently, 0, 1, 2, or 3 representing the        number of substituents on the rings, respectively, and at least        one of m or n must be ≥1;

wherein each R¹, R², R³, and R⁴ is independently selected from:

-   -   —R⁵, wherein R⁵ is selected from (C₁-C₆)alkyl, (C₂-C₆)alkenyl,        or (C₂-C₆)alkynyl; optionally substituted with 1 to 3        substituents selected from —OH, —SH, -halo, —NH₂, or NO₂;    -   —YR⁶, wherein Y is O, S, or NH; and R⁶ is selected from H or R⁵;    -   —ZR⁵, wherein Z is —N(C═O)— or —O(C═O)—;    -   -halo;    -   —NO₂;    -   —SO₃Na;    -   -azide; and    -   -glycosides    -   -and salts thereof;    -   with the proviso that the FAM is not resveratrol or        4,4′-(ethyne-1,2-diyl)diphenol (TOLCINE).

An “auxiliary autophagy modulator” or “AAM compound” or “AAM” means acompound as described herein that also has an autophagy modulatingeffect. The effect may be stimulatory or inhibitory depending on thecompound. While not intending to be bound by any particular theory, AAMcompounds may have inhibitory action by competing for a rate limitingstep, such as cellular uptake mechanisms. More specific AAM compoundsare described in a subsequent section.

Autophagy modulation refers to either up-regulation or down-regulationof the process of autophagy in the cell. Depending on the particulardisease state or condition, it may be desirable to achieve one or theother direction of regulation of autophagy, as is described later. And,referring to the discussion of hormesis, the dose of any particular FAMor AAM compound or combination or complex may achieve up-regulation ordown-regulation or both.

As used herein, the term “—(C₁-C₆)alkyl” refers to straight-chain andbranched non-cyclic saturated hydrocarbons having from 1 to 6 carbonatoms. Representative straight chain—(C₁-C₆)alkyl groups include methyl,-ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl. Representativebranched-chain —(C₁-C₆)alkyl groups include isopropyl, sec-butyl,isobutyl, tert-butyl, isopentyl, neopentyl, 1-methylbutyl,2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, and1,2-dimethylpropyl, methylpentyl, 2-methylpentyl, 3-methylpentyl,4-mehtylpentyl, 1-ethylbutyl, 2-ethylbutyl, 3-ethylbutyl,1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl,2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, and the like.More generally, the subscript refers to the number of carbon atoms inthe chain. Thus, the term “—(C₁-C₄)alkyl” refers to straight-chain andbranched non-cyclic saturated hydrocarbons having from 1 to 4 carbonatoms.

As used herein, the term “—(C₂-C₆)alkenyl” refers to straight chain andbranched non-cyclic hydrocarbons having from 2 to 6 carbon atoms andincluding at least one carbon-carbon double bond. Representativestraight chain and branched —(C₂-C₆)alkenyl groups include -vinyl,allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl,-3-methyl-1-butenyl, -2-methyl-2-butenyl, and the like.

As used herein, the term “—(C₂-C₆)alkynyl” refers to straight chain andbranched non-cyclic hydrocarbons having from 2 to 6 carbon atoms andincluding at least one carbon-carbon triple bond. Representativestraight chain and branched —(C₂-C₆)alkynyl groups include -acetylenyl,-propynyl, -1 butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl,-3-methyl-1-butynyl, -4-pentynyl, and the like.

As used herein, “—(C₁-C₁₀)alkoxy” means a straight chain or branchednon-cyclic hydrocarbon having one or more ether groups and from 1 to 10carbon atoms. Representative straight chain and branched (C₁-C₁₀)alkoxysinclude -methoxy, -ethoxy, -propoxy, -butyloxy, -pentyloxy, -hexyloxy,-heptyloxy, -methoxymethyl, -2-methoxyethyl, -5-methoxypentyl,-3-ethoxybutyl and the like.

As used herein, “—(C₁-C₆)alkoxy” means a straight chain or branchednon-cyclic hydrocarbon having one or more ether groups and from 1 to 6carbon atoms. Representative straight chain and branched (C₁-C₅)alkoxysinclude -methoxy, -ethoxy, -propoxy, -butyloxy, -pentyloxy, -hexyloxy,-methoxymethyl, -2-methoxyethyl, -5-methoxypentyl, -3-ethoxybutyl andthe like.

As used herein, the term “—(C₃-C₁₂)cycloalkyl” refers to cyclicsaturated hydrocarbon having from 3 to 12 carbon atoms. Representative(C₃-C₁₂)cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, and cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like.

As used herein, the term “—(C₄-C₁₂)cycloalkenyl” refers to a cyclichydrocarbon having from 4 to 12 carbon atoms, and including at least onecarbon-carbon double bond. Representative —(C₃-C₁₂)cycloalkenyls include-cyclobutenyl, -cyclopentenyl, -cyclopentadienyl, -cyclohexenyl,-cyclohexadienyl, -cycloheptenyl, -cycloheptadienyl, -cycloheptatrienyl,-cyclooctenyl, - cyclooctadienyl, -cyclooctatrienyl,-cyclooctatetraenyl, -cyclononenyl, -cyclononadienyl, -cyclodecenyl,-cyclodecadienyl, -norbornenyl, and the like.

As used herein a “-(7- to 12-membered)bicyclic aryl” means an bicyclicaromatic carbocyclic ring containing 7 to 12 carbon atoms.Representative -(7- to 12-membered) bicyclic aryl groups include-indenyl, -naphthyl, and the like.

As used herein a “hydroxy(C₁-C₆)alkyl” means any of the above-mentionedC₁₋₆ alkyl groups substituted by one or more hydroxy groups.Representative hydroxy(C₁-C₆)alkyl groups include hydroxymethyl,hydroxyethyl, hydroxypropyl and hydroxybutyl groups, and especiallyhydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1,2-dihydroxyethyl,2-hydroxypropyl, 3-hydroxypropyl, 3-hydroxybutyl, 4-hydroxybutyl,2-hydroxy-l-methylpropyl, and 1,3-dihydroxyprop-2-yl.

Any of the groups defined above may be optionally substituted. As usedherein, the term “optionally substituted” refers to a group that iseither unsubstituted or substituted. Optional substituents, when presentand not otherwise indicated, include 1, 2, or 3 groups eachindependently selected from the group consisting of —(C₁-C₆)alkyl, OH,halo, —C(halo)₃, —CH(halo)₂, —CH₂(halo), NH₂, —NH(C₁-C₆)alkyl, CN, SH,phenyl, benzyl, (═O), halo(C₁-C₆)alkyl-, hydroxy(C₁-C₆)alkyl-. Thus,certain substituted embodiments include those named below.

As used herein a “dihydroxy(C₁-C₆)alkyl” means any of theabove-mentioned C₁₋₆ alkyl groups substituted by two hydroxy groups.Representative dihydroxy(C₁-C₆)alkyl groups include dihydroxyethyl,dihydroxypropyl and dihydroxybutyl groups, and especially1,2-dihydroxyethyl, 1,3-dihydroxypropyl, 2,3-dihydroxypropyl,1,3-dihydroxybutyl, 1,4-dihydroxybutyl, and 1,3-dihydroxyprop-2-yl.

As used herein, the terms “halo” and “halogen” refer to fluoro, chloro,bromo or iodo.

As used herein, “—CH₂(halo)” means a methyl group where one of thehydrogens of the methyl group has been replaced with a halogen.Representative —CH₂(halo) groups include —CH₂F, —CH₂Cl, —CH₂Br, and—CH₂I.

As used herein, “—CH(halo)₂” means a methyl group where two of thehydrogens of the methyl group have been replaced with a halogen.Representative —CH(halo)₂ groups include —CHF₂, —CHCl₂, —CHBr₂, —CHBrCl,—CHClI, and —CHI₂.

As used herein, “—C(halo)₃” means a methyl group where each of thehydrogens of the methyl group has been replaced with a halogen.Representative —C(halo)₃ groups include —CF₃, —CCl₃, —CBr₃, and —CI₃.

As used herein, “(halo)_(p)(C₁-C₆)alkyl-” means a (C₁-C₆)alkyl chainsubstituted with halo in p locations, where p is 1, 2 or 3. The halosubstituents may be substituted on the same or a different carbon in the(C₁-C₆)alkyl. Representative “(halo)_(p)(C₁-C₆)alkyl-” groups include,for example —CH₂CHF₂, —CH₂CH₂CH₂Cl, —CHBr₂, —CHBrCl, —CHClI, —CH₂CHI₂,—CH₂CH₂CHClCH₂Br, and the like.

As used herein, “azide” means a substituent of the formula —N═N═N.

As used herein, “glycoside” means a 5- or 6-membered cyclic sugarconnected to the compound of Formula I. The bond is generally aglycosidic bond from an anomeric carbon of the sugar, and may be madevia: (1) an oxygen atom, thus forming an “O-glycoside”, (2) a nitrogenatom, thus forming an “N-glycoside” or (3) a sulfur atom, thus formingan “S-glycoside.” Glycosides may be mono- or di-saccharides, having oneor two ring structures. Representative glycosides formed from 6 memberedsugars glucosides, galactosides, mannosides, and altrosides; andglycosides formed from the 5 membered sugars, include ribosides,arabinosides, xylosides and lyxosidees. The sugars may contain optionalsubstituents, but many embodiments contain only the native —H and —OHsubstituents that define the respective sugars.

Overlap exists in the literature among use of the terms “wound,”“ulcer,” and “sore” and, furthermore, the terms are often used atrandom. Therefore, in the present context the term “wounds” encompassesthe term “ulcer”, “lesion”, “sore” and “infarction”, and the terms areused interchangeably unless otherwise indicated. Wounds have beenclassified by many criteria, including size or area (large or small),depth or layer involvement, causation, difficulty in healing, etc. Someclassify wounds by i) small tissue loss due to surgical incisions, minorabrasions and minor bites, or as ii) significant tissue loss, such as inischemic ulcers, pressure sores, fistulae, lacerations, severe bites,thermal burns and donor site wounds (in soft and hard tissues) andinfarctions. All types and classification of wounds are encompassed bythe invention.

The term “skin” is used in a very broad sense embracing the epidermallayer of the integument and, in those cases where the skin surface ismore or less injured, also the dermal layer beneath. Apart from thestratum corneum, the epidermal layer of the skin is the outer(epithelial) layer and the deeper connective tissue layer of the skin iscalled the “dermis,” which contains the nerves and terminal organs ofsensation.

A method that “promotes the healing of a wound” results in the woundhealing more quickly as a result of the treatment than a similar woundheals in the absence of the treatment. “Promotion of wound healing” canalso mean that the method regulates the proliferation and/or growth of,inter alia, keratinocytes, or that the wound heals with less scarring,less wound contraction, less collagen deposition and more superficialsurface area. In certain instances, “promotion of wound healing” canalso mean that certain methods of wound healing have improved successrates, (e.g., the take rates of skin grafts) when used together with themethod of the present invention.

The phenomenon of hormesis is commonly associated with compounds thatinduce biologically opposite effects in a dose dependent fashion, and iswell described in the literature, for example: Calabrese E J, Bachmann KA, Bailer A J, Bolger P M, Borak J, et al. (2007), Biological stressresponse terminology: Integrating the concepts of adaptive response andpreconditioning stress within a hormetic dose-response framework.Toxicol Appl Pharmacol. 222:122-128; Penniston K L, Tanumihardjo S A.The acute and chronic toxic effects of vitamin A. Am J Clin Nutr. 2006;83:191-201; and Cook R, Calabrese E J. The importance of hormesis topublic health. Cien saude Colet. 2007; 12:955-963. Commonly there is astimulatory or beneficial effect at low doses and an inhibitory or toxiceffect at high doses. Hormesis has also been characterized asautoprotection, adaptive response, and preconditioning, among others;and by the shape of its dose response curve including: e.g. β-curve,biphasic, bell-shaped, U-shaped or inverted-U shaped, bimodal,functional antagonism, and dual response, among others. Known hormeticsubstances (or “hormetins”) include: vitamin A, ferulic acid, chalcone,rapamycin, epigallocatechin-3-gallate and many others.

Hormesis can also represent adaptive responses where a moderate stressis applied in order to provide the organism with adaptive resistancewhen faced with a severe stressor. Environmental stresses such asoxidative metabolic and thermal stress serve as hormetins used to inducea specific response or adaptive change (Mattson M P. Hormesis defined,Ageing Res Rev. 2008; 7:1-7). Hormetins have been shown to activatevarious stress and detoxification pathways including; heat shockproteins, antioxidant, protein chaperones, metabolism, calciumhomeostasis and growth factors (Mattson M P, Cheng A. Neurohormeticphytochemicals: Low-dose toxins that induce adaptive neuronal stressresponses. Trends Neurosc. 2006; 29:632-639; and Mattson, 2008, notedabove). These dose response effects have been analyzed for a range ofnatural signaling molecules including nitric oxide, adenosine, opioids,adrenergic agents, prostaglandins, estrogens, androgens,5-hydroxytryptamine and dopamine (Calabrese E J, Bachmann K A, Bailer AJ, Bolger P M, Borak J, et al. (2007), Biological stress responseterminology: Integrating the concepts of adaptive response andpreconditioning stress within a hormetic dose-response framework.Toxicol Appl Pharmacol. 222:122-128 and Hayes D P. Nutritional hormesis.Eur J Clin Nutr. 2007; 61:147-159) Many of these hormetics areconcentrated by bacteria, fungi, viruses and plants to protectthemselves from predatory species. However, when many of thesesubstances are utilized in lower concentrations they can have beneficialeffects.

A hydrogel is a dilute cross-linked aqueous system comprising water anda gelling agent such as a polymeric plastic or polysaccharide which canabsorb and retain significant amounts of water to form three-dimensionalnetwork structures. The hydrogel structure is created by the interactionof water with hydrophilic groups or domains present in the polymericnetwork upon hydration. Hydrogels are categorized principally as weak orstrong depending on their flow behavior in steady-state.

Gelation occurs when the polymer concentration increases, and dispersespolymers begin to branch and form crosslinks. Once a criticalconcentration is reached the sol becomes a gel and the sol-geltransition occurs. Gels may be considered chemically linked orphysically linked. Physical gels can be subcategorized as strong orweak, depending on the nature of the bonds, with strong physicalapproaching chemical gels in linkage permanence. Strong physical bondsinclude: lamellar microcrystals, glassy nodules or double and triplehelices; whereas weak physical gels include: hydrogen bonds, blockcopolymers, micelles, and ionic associations.

Hydrogels, due to their significant water content possess a degree offlexibility similar to natural tissue, and may exhibit viscoelastic orpure elastic behavior, and stickiness. Properties of a hydrogel may bemodified by controlling the polarity, surface properties, mechanicalproperties, and swelling behavior. Gels may exhibit significant volumechanges in response to small changes in pH, ionic strength, temperature,electric field, and light. Biodegradable hydrogels, containing labilebonds, are advantageous in applications such as tissue engineering,wound healing and drug delivery. These labile bonds can be presenteither in the polymer backbone or in the cross-links used to prepare thehydrogel. The labile bonds can be broken under physiological conditionseither enzymatically or chemically, in most of the cases by hydrolysis(Bajpai, A. K., Shukla, S. K., Bhanu, S. & Kankane, S. (2008).Responsive polymers in controlled drug delivery. Progress in PolymerScience. 33: 1088-1118).

Ionic polymers having negatively charged groups at physiological pH canbe cross-linked by the addition of multivalent cations; and evenmonovalent cations (e.g. K³⁰ , Na⁺, etc.) may shield or screen therepulsion of negatively charged groups (e.g. SO⁻ ₃) to form stable gels.Examples include: Na⁺alginate⁻; chitosan-polylysine; chitosan-glycerolphosphate salt (Syed K. H. Gulrez¹ and Saphwan Al-Assaf. Progress inmolecular and environmental bioengineering from analysis and modeling totechnology applications Chapter 5: Hydrogels: Methods of preparation,characterization and applications. Publisher InTech. Aug. 1, 2011(34))and chitosan-dextran hydrogels (Hennink, W. E. & Nostrum, C. F. (2002).Novel crosslinking methods to design hydrogels. Advanced drug deliveryreviews. 54: 13).

Alginate is a naturally occurring anionic polysaccharide typicallyobtained from brown seaweed, and has been extensively investigated andused for many biomedical applications, due to its biocompatibility, lowtoxicity, relatively low cost, and ability to form hydrogels by additionof divalent cations such as Ca²⁺. Alginate hydrogels have been used in avariety of applications including; wound healing and drug delivery.Alginate wound dressings maintain a moist wound environment, minimizebacterial infection at the wound site, and facilitate wound healing.Drug molecules can be released from alginate gels in a controlledmanner, depending on the cross-linker and cross-linking methodsemployed. In addition, alginate gels can be orally administrated orinjected, making them extensively useful in the pharmaceutical arena.

Alginates are polysaccharides constituted by variable amounts ofβ-D-mannuronic acid and its C5-epimer α-L-guluronic acid linked by 1-4glycosidic bonds. The capability of alginate to confer viscosity insol-gels is dependent on its molecular mass (MM). The molecular mass(MM) of algal alginates has been found to range from 48 to 186 kDa (38);whereas some alginates isolated from A. vinelandii present MM in therange of 80 to 4,000 kDa (Galindo, E.; Peña, C. ; Núñez, C.; Segura, D.& Espin, G. (2007). Molecular and bioengineering strategies to improvealginate and polyhydroxyalkanoate production by Azotobacter vinelandii.Microbial Cell Factories, 6, 1-1). The saccharide monomers aredistributed in blocks of continuous mannuronate residues (M), guluronateresidues (G) or alternating residues (MG), depending on the sourcespecies Smidsrod, O. & Draget, K. (1996). Chemistry and physicalproperties of alginates.Carbohydrates European, 14, 6-12). The G-blocksof alginates participate in intermolecular cross-linking with divalentcations (e.g., Ca²⁺) to form hydrogels. The composition (i.e., M/Gratio), sequence, G-block length, and molecular weight are criticalfactors which alter the physical properties (e.g. increasing G-blocklength increases ionic binding and the mechanical rigidity of the gel)of alginate and alginate hydrogels. These same properties control thestability of the gels along with the release rate of alginatescontaining drugs. Alginates with a low M/G ratio form strong and brittlegels, while alginates with a high M/G ratio form weaker and softer, butmore elastic gels. Finally, bacterial alginates are acetylated to avariable extent at positions O-2 and/or O-3 of the mannuronate residues(Skjak-Braek, G.; Grasdalen, H. & Larsen, B. (1986). Monomer sequenceand acetylation pattern in some bacterial alginates. CarbohydratesResearch, 154, 239-250). The variability in molecular mass, monomerblock structure and acetylation all influence the physicochemical andrheological characteristics of the gel polymer.

The majority of alginates use the divalent cation calcium or monovalentions such as Na⁺ or K⁺ while other ions such as Mg²⁺ have been proposedbut for the gelation process to occur the concentration of magnesiumions required is 5-10 times higher than that of calcium (Topuz, F.,Henke, A., Richtering, W. & Groll, J. (2012). Magnesium ions andalginate do form hydrogels: a rheological study. Soft Matter.8:4877-4881). Alginates may have decreased water solubility such asalginic acid or calcium alginate where the ion is shielded fromionization resulting in insoluble alginates. Water soluble alginates canbe made simply by creating salts using monovalent anions such as Na⁺ orK⁺ or a non polar group such as NH₄ which is generally water soluble.

First Autophagy Modulators (FAMs)

As noted, the first autophagy modulators are compounds of Formula I,which comprises two phenyl rings joined by a linker, L, and having atleast one R¹ or R² attached to a phenyl ring. In some embodiments, L is—C≡C— and these FAM compounds are known as “tolans,” which are generallylinear from phenyl ring to phenyl ring. In other embodiments, L is—CH═CH— and these FAM compounds are known as “stilbenes” which areisomeric in cis and trans forms about the double bond. In someembodiments the FAM compound is a trans stilbene. In still otherembodiments, L is —CR_(a)═CR_(b)— where R_(a) and/or R_(b) may be aphenyl ring or H. These are also stilbenes, in this case “phenylstilbene derivatives” and they may also be trans stilbenes or cisstilbenes.

There are two “primary” phenyl rings shown in the structure andcontaining substituents R¹ and R², of which at least one must be present(i.e. at least one of m or n is ≥1). Optionally, there are also up totwo “secondary” phenyl rings within the options for R_(a) and R_(b). Oneach phenyl ring (up to four possible, two primary and two secondary)there may be from zero to five of each substituent R¹⁻⁴. In certainembodiments, there are from one to three R¹, and/or from one to three R²substituents on the primary phenyl rings. In some embodiments, theposition of R¹ and/or R² on the primary phenyl rings is mostly at thepara and meta positions, namely the 3, 4 or 5 position on one phenylring and the 3′, 4′ and 5′ positions on the other phenyl ring, althoughit is also possible to have substituents in the ortho position (2, 2′, 6and 6′). There may be one, two or three R¹ substituents on the firstphenyl ring, and correspondingly, from zero to three R² substituents onthe second phenyl ring. Conversely, there may be one, two or three R²substituents on the second phenyl ring, and correspondingly, from zeroto three R¹ substituents on the first phenyl ring. Additionally, in someembodiments, the secondary phenyl rings may contain one to threesubstituents R³ and R⁴. All permutations within these are possible, forexample: one R¹ and one R²; two R¹ and two R²; three R¹ and three R²;one R¹ and two R²; one R¹ and three R²; two R¹ and one R²; two R¹ andtwo R²; two R¹ and three R²; three R¹ and one R²; or three R¹ and twoR². Likewise with R³ and R⁴. Each R¹⁻⁴ is independently selected and iftwo or more are present they may be the same or different. Examples ofsome specific first autophagy modulators are given in Table A, thetolans being analogues of the stilbenes so detailed structures are notnecessary for each individual compound.

TABLE A Certain Representative FAM compounds The nature and position(s)of R¹ and R² substituents Stilbenes Tolans

Hydroxy 3,5-dihydroxy-trans-stilbene (aka, Pinosylvin);3,5-dihydroxytolan, 3,4-dihydroxy-trans-stilbene, 3,4-dihydroxytolan,3,4,5-trihydroxy-trans-stilbene, 3,4,5-trihydroxytolan,

3,3′,4,5′-tetrahydroxy-trans-stilbene (aka piceatannol); and3,3′,4,5′-tetrahydroxytolan; and 3,3′,4,4′-tetrahydroxy-trans-stilbene3,3′,4,4′-tetrahydroxytolan 3,4,4′-trihydroxy-trans-stilbene;3,5,4′-trihydroxytolan 3,3′,4′-trihydroxy-trans-stilbene;3,4,4′-trihydroxytolan; 4,4′-dihydroxy-trans-stilbene;3,3′,4′-trihydroxytolan; 2,4,4′-trihydroxy-trans-stilbene;2,4,4′-trihydroxytolan; 2,4,2′,4′-tetrahydroxy-trans-stilbene2,4,2′,4′-tetrahydroxytolan Alkyl and 3,5-dihydroxy-4-ethylstilbene,3,5-dihydroxy-4-ethyltolan, mixed 3,5-dihydroxy-4′-ethylstilbene;3,5-dihydroxy-4′-ethyltolan 3,5-dihydroxy-4-isopropyl-trans-stilbene;3,5-dihydroxy-4-isopropyltolan; and3,5-dihydroxy-4-isopropyl-trans-4′-hydroxystilbene;3,5-dihydroxy-4-isopropyl-trans-4′hydroxytolan;3,5-dihydroxy-4-isopropyl-trans-3′,4′-dihydroxystilbene;3,5-dihydroxy-4-isopropyl-trans-3′,4′-dihydroxytolan;3,5-dihydroxy-4-isopropyl-trans-3′,5′-dihydroxystilbene;3,5-dihydroxy-4-isopropyl-trans-3′,5′-dihydroxytolan;3,5-dihydroxy-4-isopropyl-trans-3′,4′,5′-trihydroxystilbene3,5-dihydroxy-4-isopropyl-trans-3′,4′,5′-trihydroxytolan3,4-dihydroxy-4′-isopropyl-trans-stilbene;3,4-dihydroxy-4′-isopropyltolan;3,5-dihydroxy-4′-isopropyl-trans-stilbene;3,5-dihydroxy-4′-isopropyltolan;3,4,5-trihydroxy-4′-isopropyl-trans-stilbene;3,4,5-trihydroxy-4′-isopropyltolan;4-hydroxy-4′-isopropyl-trans-stilbene; 4-hydroxy-4′-isopropyltolan4′-hydroxy-3,4-dimethyl-trans-stilbene, 4′-hydroxy-3,4-dimethyltolan,4′-hydroxy-3,4,5-trimethyl-trans-stilbene,4′-hydroxy-3,4,5-trimethyltolan, 4′-hydroxy-4,5-dimethyl-trans-stilbene4′-hydroxy-4,5-dimethyltolan. Glycosides and mixed

E.g. E.g. 3,5′-dihydroxy-4′-methoxystilbene-5-O-β-D-glucoside;3,5′-dihydroxy-4′-methoxytolan-5-O-β-D-glucoside;3′,4′-dihydroxy-3-methoxystilbene 5-O-β-D-glucoside;3′,4′-dihydroxy-3-methoxytolan-5-O-β-D-glucoside; 3,4′-dihydroxystilbene5-O-β-D-glucoside; 3,4′-dihydroxytolan 5-O-β-D-glucoside;2,3-dihydroxy-4′-methoxystilbene 5-O-β-D-glucoside;2,3-dihydroxy-4′-methoxytolan 5-O-β-D-glucoside;2,3,3′-trihydroxy-4′′-methoxystilbene 5-O-β-D-glucoside2,3,3′-trihydroxy-4′-methoxytolan 5-O-β-D-glucoside; Halo and mixed

3,5-dihydroxy-4′-chlorostilbene, 3,5-dihydroxy-4′-chlorotolan,3,4-dihydroxy-4′-chlorostilbene, or 3,4-dihydroxy-4′-chlorotolan, or4,5-dihydroxy-4′-chlorostilbene, 4,5-dihydroxy-4′-chlorotolan,3,4,5-dihydroxy-4′-chlorostilbene, and 3,4,5-dihydroxy-4′-chlorotolan2,3,4,5,6-penthydroxy-4′-chlorostilbene 3,4-dihydroxy-4′-fluorostilbene,or 3,4-dihydroxy-4′-fluorotolan or 4,5-dihydroxy-4′-fluorostilbene,4,5-dihydroxy-4′-fluorotolan, 3,4,5-trihydroxy-4′-fluorostilbene,3,4,5-trihydroxy-4′-fluorotolan, 3,5-dihydroxy-4′-fluorostilbene,3,5-dihydroxy-4′-fluorotolan

3,4-dihydroxy-4′-(trifluoro)methylstilbene3,4-dihydroxy-4′-(trifluoro)methyltolan Thiol and3,5-dihydroxy-4′-thiolstilbene, 3,5-dihydroxy-4′-thioltolan, mixed3,4-dihydroxy-4′-thiolstilbene, or 3,4-dihydroxy-4′-thioltolan, or4,5-dihydroxy-4′-thiolstilbene, 4,5-dihydroxy-4′-thioltolan,3,4,5-dihydroxy-4′-thiolstilbene, 3,4,5-dihydroxy-4′-thioltolan,3,4′-dithiol-trans-stilbene, 3,4′-dithiol-tolan,4,4′-dithiol-trans-stilbene, 4,4′-dithiol-tolan, Alkoxy (O- alkyl andthioether (S-alkyl) and mixed

3,5,4′-trimethoxy-trans-stilbene, 3,5,4′-trimethoxytolan,3,4,4′-trimethoxy-trans-stilbene, or 3,4,4′-trimethoxytolan, or4,5,4′-trimethoxy-trans-stilbene, 4,5,4′-trimethoxytolan,3,4,5,4′-tetramethoxy-trans-stilbene, 3,4,5,4′-tetramethoxytolan,4′-hydroxy-3,5-dimethoxy-trans-stilbene, 4′-hydroxy-3,5-dimethoxytolan,4′-hydroxy-3,4-dimethox-trans-stilbene, or4′-hydroxy-3,4-dimethoxytolan, or4′-hydroxy-4,5-dimethox-trans-stilbene, 4′-hydroxy-4,5-dimethoxytolan,4′-hydroxy-3,4,5-trimethox-trans-stilbene,4′-hydroxy-3,4,5-trimethoxytolan,3,5-dihydroxy-4′-methoxy-trans-stilbene, 3,5-dihydroxy-4′-methoxytolan,4,4′-dimethoxy-trans-stilbene, 4,4′-dimethoxytolan,3,5-dimethoxy-4′-thiomethyl-trans-stilbene3,5-dimethoxymethoxy-4′-thiomethyltolan3,5,3′-trihydroxy-4′-methoxy-trans-stilbene3,5,3′-trihydroxy-4′-methoxytolan,4,4′-dihydroxy-3-methoxy-trans-stilbene 4,4′-dihydroxy-3-methoxytolan3,4-dihydroxy-4′-methoxy-trans-stilbene 3,4-dihydroxy-4′-methoxytolan3,4-dimethoxy-trans-stilbene, 3,4-dimethoxytolan,3,4′-dimethoxy-trans-stilbene, 3,4′-dimethoxytolan, Acyl and4′hydroxy-5-acetoxy-trans-stilbene, 4′hydroxy-5-acetoxy-tolan, mixed3,4′-dihydroxy-5-acetoxy-trans-stilbene, 3,4′-dihydroxy-5-acetoxy-tolan,3,5-dihydroxy-4′-acetoxy-trans-stilbene, 3,5-dihydroxy-4′-acetoxy-tolan,3,4′-dihydroxy-4-acetoxy-trans-stilbene, 3,4′-dihydroxy-4-acetoxytolan,4,4′-dihydroxy-3-acetoxy-trans-stilbene, 4,4′-dihydroxy-3-acetoxytolan,3,4-dihydroxy-4′-acetoxy-trans-stilbene 3,4-dihydroxy-4′-acetoxytolan3,4,3′-trihydroxy-4′-acetoxy-trans-stilbene,3,4,3′-trihydroxy-4′-acetoxytolan,3,4,4′-trihydroxy-3′-acetoxy-trans-stilbene,3,4,4′-trihydroxy-3′-acetoxytolan,3,5,4′-trihydroxy-3′-acetoxy-trans-stilbene,3,5,4′-trihydroxy-3′-acetoxytolan,3,5,3′-trihydroxy-4′-acetoxy-trans-stilbene,3,5,3′-trihydroxy-4′-acetoxytolan Amide and mixed

3,5-dihydroxy-4′-acetamide-trans-stilbene,3,5-dihydroxy-4′-acetamidetolan,4,5-dihydyroxy-4′-acetamide-trans-stilbene, or4,5-dihydyroxy-4′-acetamidetolan, or3,4-dihydyroxy-4′-acetamide-trans-stilbene,3,4-dihydyroxy-4′-acetamidetolan,3,4,5-trihydyroxy-4′-acetamide-trans-stilbene,3,4,5-trihydyroxy-4′-acetamidetolan,3,4,3′-trihydyroxy-4′-acetamide-trans-stilbene,3,4,3′-trihydyroxy-4′-acetamidetolan,3,4,5′-trihydyroxy-4′-acetamide-trans-stilbene,3,4,5′-trihydyroxy-4′-acetamidetolan,4′hydroxy-5-acetamide-trans-stilbene, 4′-hydroxy-5-acetamidetolan,3,4′-dihydroxy-5-acetamide-trans-stilbene,3,4′-dihydroxy-5-acetamidetolan,4,4′-dihydroxy-3-acetamide-trans-stilbene,4,4′-dihydroxy-3-acetamidetolan,3,4-dihydroxy-4′-acetamide-trans-stilbene,3,4-dihydroxy-4′-acetamidetolan,3,4′-dihydroxy-4-acetamide-trans-stilbene,3,4′-dihydroxy-4-acetamidetolan,4,4′-dihydroxy-3-acetamide-trans-stilbene,4,4′-dihydroxy-3-acetamidetolan,3,4-dihydroxy-4′-acetamide-trans-stilbene3,4-dihydroxy-4′-acetamidetolan,3,4,4′-trihydroxy-3′-acetamide-trans-stilbene,3,4,4′-trihydroxy-3′-acetamidetolan,3,5,4′-trihydroxy-4-acetamide-trans-stilbene,3,5,4′-trihydroxy-4-acetamidetolan,3,4,3′-trihydroxy-4′-acetamide-trans-stilbene,3,4,3′-trihydroxy-4′-acetamidetolan,3,4,4′-trihydroxy-3′-acetamide-trans-stilbene,3,4,4′-trihydroxy-3′acetamidetolan,3,3′-dihydroxy-4′-acetamide-trans-stilbene,3,3′-dihydroxy-4′-acetamidetolan, Azides

4-hydroxy-4′-azidostilbene, 4-hydroxy-4′-azidotolan,3,5-dihydroxy-4′-azidostilbene, 3,5-dihydroxy-4′-azidotolan,4,5-dihydroxy-4′-azidostilbene, or 4,5-dihydroxy-4′-azidotolan, or3,4-dihydroxy-4′-azidostilbene, 3,4-dihydroxy-4′-azidotolan,3,4,5-trihydroxy-4′-azidostilbene, 3,4,5-trihydroxy-4′-azidotolan, Misc.

4-hydroxy-4′-nitro-trans-stilbene, 4-hydroxy-4′-nitro-tolan,

3,5-dihydroxy-4′-nitro-trans-stilbene, 3,5-dihydroxy-4′-nitro-tolan,

3,4-dihydroxy-4′-nitro-trans-stilbene, or 3,4-dihydroxy-4′-nitro-tolan,or 4,5-dihydroxy-4′-nitro-trans-stilbene, 4,5-dihydroxy-4′-nitro-tolan,

3,4,5-trihydroxy-4′-nitro-trans-stilbene3,4,5-trihydroxy-4′-nitro-tolan,

Many of the stilbene compounds are well studied, naturally occurringmolecules and some are readily commercially available. Others may besynthesized by routine methods, such as those described by: Ali, M. A.,Kondo, K. and Tsuda, Y. (1992). Synthesis and Nematocidal activity ofHydroxystilbenes. Chem. Pharm. Bull. 40(5):1130-1136; and Thakkar, K.,Geahlen, R. L. and Cushman, M. (1993). Synthesis and Protein-tyrosinekinase inhibitory activity of polyhydroxylated stilbene analogues ofpiceatannol. J. Med. Chem. 36: 2950-2955). Phenyl stilbene derivatives,i.e. those in which at least one R_(a) or R_(b) is a phenyl ring, andmany other stilbenes may be synthesized according to the methods shownin the dissertation of Zhenlin Bai, Substituted Stilbenes and1,2-Diaryl-1,2-diazidoethanes as Potential Anticancer Agents: Synthesesand Estrogenic/Antiestrogenic Properties in MCF-7-2a Cells, imFachbereich Biologie, Chemie, Pharmazie der Freien Universitat Berlin(2006). Glucoside derivatives may be obtained according to proceduresdescribed in WO2007/020673 A1. Tolans may be synthesized using thegeneral procedures described in U.S. Pat. No. 6,599,945 B2 of Docherty &Tsai.

It can be noted that the FAM compounds described above are generallypolar, and have certain electronegative substituents (e.g. —OH, —OCH₃,—NO₂, -halo, —O(C═O)R, etc.) at the respective ends. While this is notdeemed essential, it may be desirable to provide for a liquidcrystal-like behavior for molecules to assume a lyotrophic or partiallyordered structure in solution state.

FAM compounds also include salts of the compounds identified above. FAMcompounds, especially those mono or poly-hydroxylated compounds, easilyrelease one or more protons depending on pH to form anions. Such anionsmay be combined with cations, such as the mono-, di-, and tri-valaentcations to form salts. For monovalent cations (M+) a single FAM islinked to form M⁺FAM⁻ salts. Similarly, for a divalent cation (M²⁺) twoFAM molecules are linked to form M²⁺(FAM⁻)₂ salts; and for a trivalentcation (M³⁺) three FAM molecules are linked to form M⁺³(FAM⁻)₃ salts.The salts are often readily soluble in aqueous media, which mayfacilitate formulations. Illustrative, but not limiting, cations for FAMsalt formation include: Na⁺ or K⁺, Mg²⁺, Mn²⁺, Zn²⁺, Ca²⁺, Cu⁺,Cu²⁺Fe²⁺, and Fe³⁺.

Auxiliary Autophagy Modulators (AAMs) and Formulations

In certain embodiments, the FAM is used in combination with an auxiliaryautophagy modulator (AAM). The AMM, when used, may take any of severalforms described herein, falling into any of the following classes:vitamins, amino acids, acidic sugars, and quinine derivatives

Vitamins

Vitamins A, B, C, D, E, and K may all be useful as AAMs. By convention,the term “vitamin” includes neither other essential nutrients, such asdietary minerals, essential fatty acids, or essential amino acids (whichare needed in greater amounts than vitamins), nor the great number ofother nutrients that promote health. Thirteen vitamins are universallyrecognized at present. Vitamins are classified by their biological andchemical activity, not their structure. Thus, each “vitamin” refers to anumber of “vitamer” compounds that all show the biological activityassociated with a particular vitamin. Such a set of chemicals is groupedunder an alphabetized vitamin “generic descriptor” title, such as“vitamin A”, which includes multiple compounds as described below.Vitamers by definition are convertible to the active form of the vitaminin the body, and are sometimes inter-convertible to one another, aswell. In certain embodiments the salt forms of the vitamins, generallywithout long side aliphatic chains, are excellent AAMs. In certainembodiments, the oxygen containing vitamins are suitable AAMS.

Most vitamins will also form salts that are also within the scope ofAAMs. For example, vitamins may combine to form salts with cationicelements like sodium, potassium, magnesium, manganese, calcium, copper,zinc, or iron. Additionally vitamins can form a diethanolamine salt, a2-amino-2-ethyl-1,3-propanediol salt, a triethanolamine salt, amorpholine salt, a piperazine salt, a piperidine salt, an arginine salt,a lysine salt and a histidine salt. Some vitamins form acetates,palmitates, oleates, linoleates, stearates, lactates, succinates,maleates, citrates, and the like.

Vitamin A refers to a group of lipid soluble, unsaturated, isoprenoidcompounds that includes but is not limited to retinol, retinal, retinoicacid, carotenoids, retinyl acetate, retinyl palmitate, α-carotene,β-carotene, γ-carotene, β-cryptoxanthin, xanthophyll, crytoxanthin,13-cis retinoic acid, 13-trans retinoic acid, tretinoin, ATRA (all transretinoic acid), lutin, 11-cis-retinal, 11-cis-retinol, 9-cis-retinal,Lecithin, retinyl esters, 9-cis-β-carotene, retinyl palmitate,Acitretin, Vitamin A₂ (3,4-dehydroretinol), A₃(3-hydroxyretinol), andsalts thereof. All isomeric and stereochemical forms of theseisoprenoids are encompassed in the invention.

Vitamin B includes but is not limited to the following compounds:

thiamine (B1); riboflavin (B2); niacin or niacinamide (forms of B3);pantothenic acid, panthenol, pantothenol and calcium pantothenate (formsof B5); pyridoxine, pyridoxine 5′-phosphate, pyridoxal, pyridoxalphosphate, pyridoxal 5′-phosphate, pyridoxamine, pyridoxamine5′-phosphate, 4-pyridoxic acid (forms of B6); biotin, vitamin H orcoenzyme R (Forms of B7); Folic Acid, folate, vitamin M, vitamin Bc,pteroyl-L-glutamic acid and pteroyl-L-glutamate, (forms of B9); andCobalamin, Cyanocobalamin, Hydroxycobalamin, Methylcobalamin,Adenoxylcobalamin (forms of B12); and salts thereof.

Vitamin C refers to ascorbic acid, its anion ascorbate, and salts ofascorbate, as well as ascorbyl palmitate and salts thereof (e.g.ascorbyl palmitate, magnesium ascorbyl palmitate, manganese ascorbylpalmitate, calcium ascorbyl palmitate, zinc ascorbyl palmitate, ironascorbyl palmitate), benzyl ascorbate, and 2-ascorbyl phosphate.

Vitamin D refers to a group of lipid soluble secosteroid molecules andincludes but is not limited to: calcidiol, calcifero (INN),Ergocalciferol and lumisterol (forms of D1); ergocalciferol,fromergosterol, and 25-hydroxy vitamin D₂ (forms of D2);Cholecalciferol, 7-dehydrocholesterol, and 25-hydroxycholecalciferol (or25-hydroxyvitamin D₃, abbreviated 25(OH)D₃, (forms of D3);22-dihydroergocalciferol (D4); Sitocalciferol, 7-dehydrositosterol (D5);25-D-glucuronic acid, 25-D-hexuronic acid, 25-hydroxy vitaminD₂-25-β-D-glucuronide, and salts thereof.

Vitamin E refers to a group of lipid soluble compounds that are eithertocopherols or tocotrienols, the most active of which is α-tocopherol.Other tocopherols include, beta, gamma, delta. Similarly, tocotrienolsexist in alpha, beta, gamma and delta forms as well. All isomeric andstereochemical forms of these tocopherols and tocotrienols and theirsalts are encompassed in the invention. For example, synthetic vitamin Eis a mixture of eight isomeric forms, usually labeled “all-rac” or “dl.”Tocopherol and tocotrienol derivatives include all R and all Sstereoisomers of tocopherols (RRR, RRS, RSR, SRR, RSS, SRS and SSS) andthe two stereoisomers of tocotrienols (e.g. R or S-α-tocotrienols).Other examples include: Conjugated vitamin E molecules; vitamin E ortocopherol or tocotrienol esters; alpha-tocopheryl acetate; vitamin Eesters (e.g. alpha-tocopheryl succinate) include a group of compoundsformed by esterifying a vitamin E molecule with a carboxylic acid;d-α-tocopherol is often a mixture of two or more enantiomers of othertocopherols (β,γ,δ,ε,ζ,η) or as tocotrienols, n-propionate or linoleatesuch as vitamin E acetate or alpha-tocopheryl acetate. Water solubleforms of vitamin E include: Magnesium R-(+)-alpha lipoate,6-hydroxy-2,5,7,8-tramethylchroman-2-carboxylic acid (trolox), or saltsof vitamin E

Vitamin K refers to a group of compounds having a2-methyl-1,4,naphthoquinone core and a side chain at the 3 position.Vitamers K₁ (phylloquinone, phytomenadione, or phytonadione) and K₂(menaquinones) are naturally occurring. In fact, K₂ is not one, but aseries of compounds having varying-length isoprenoid side chains; andthe menaquinone family is sometimes designated MK-n, where n is thenumber of isoprenoid groups, n=4 being the most common. In addition,some synthetic vitamin K analogs have been made, including K₃(menadione) which has no side chain, K₄, K₅ (2-methyl-4-amino-1-naphtholhydrochloride), vitamin K₆ (2-methyl-1,4-naphthalenediamine) and K₇.Many vitamin K compounds form salts and the divalent salts are mostuseful as AAMs. For example, salts of a cations may take the form:M(Ki)₂ where M is a divalent cation or M(Ki)₃ where M is a trivalentcation. In certain embodiments, useful AAMs include salt dimers ofvitamin K and a divalent cation like Ca or Mg.

Vitamin P (although a somewhat outdated term) refers to a group offlavonoids, having the general structure of a 15-carbon skeleton, whichconsists of two phenyl rings (A and B) and heterocyclic ring (C). Thiscarbon structure can be abbreviated C6-C3-C6. Based on the nature of thesubstituents and position on the skeleton, flavonoids fall into one ofthree chemical classes: (1) flavonoids or bioflavonoids based on theflavone core (2-phenyl-1,4-benzopyrone); (2) isoflavonoids based on thea 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) core structure; and(3) neoflavonoids, based on a 4-phenylcoumarine(4-phenyl-1,2-benzopyrone) core structure.

Amino Acids

Certain amino acids and their derivatives are also useful as AAMs. As iswell known, and amino acid has the general formula

where R is any of several well understood side chains. There are 20amino acids coded by generic codes and humans synthesize 11 of these,making the other 9 “essential” amino acids, which must be consumed inthe diet. The 20 are: alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. All isomeric and stereochemical formsof these amino acids, and salts of these amino acids, are encompassed inthe invention. In certain embodiments, useful amino acids include butare not limited to: tyrosine, phenylalanine, cysteine, serine,threonine, and tryptophan. Additionally certain amino acid derivativesare useful, for example lycopene and N-actylcysteine (NAC)

Acidic Sugars

Acidic sugars include mono-, and di-saccharides formed from 4 to6-member aldoses and ketoses. They are generally acidic due to thereadiness with which a proton is released from the many hydroxyl groups.Useful monosaccharides, include but are not limited to erythrose,erythulose, threose, ribose, ribulose, arabinose, xylose, xylulose,glucose, dextrose (or D-glucose), mannose, glactose, fructose, andsorbose. Useful disaccharides include but are not limited to maltose,sucrose, lactose, cellobiose and trehalose. All isomeric andstereochemical forms of these sugars are encompassed in the invention.

Quinone Derivatives

Quinone derivatives include those having 1, 2 or three rings, thereforeincluding 1,4-benzoquinones based on

1,4-naphthoquinones based on

9,10-anthraquinones based on

and 1,3 indandiones based on

These quinone derivatives may contain substituents at any position otherthan the ketones, the substituents generally being selected fromhydroxyl, methoxy, methyl, ethyl, halo, and amino. From1-4 hydroxylsubstituents are particularly useful. For example, other examples ofhydroxy-1,4-benzoquinone derivatives include 2-hydroxy-1,4-benzoquinone,2,3-dihydroxy-1,4-benzoquinone, 2,5-dihydroxy-1,4-benzoquinone,2,6-dihydroxy-1,4-benzoquinone, 2,3,5-trihydroxy-1,4-benzoquinone, and2,3,5,6-Tetrahydroxy-1,4-benzoquinone. Other 1,4-benzoquinonederivatives include: 2,6-Dimethoxy-1,4-benzoquinone,2,3,5,6-Tetramethyl-1,4-benzoquinone, 1,4-benzoquinonetetracarboxylicacid, blatellaquinone, 2,5-Dichloro-3,6-dihydroxybenzoquinone(chloranilic acid), and 2-Isopropyl-5-methylbenzo-1,4-quinone(thymoquinone).

Examples of mono-, di-, and tetra-hydroxy-1,4-naphthoquinones include2-hydroxy-1,4-naphthoquinone (lawsone), 5-hydroxy-1,4-naphthoquinone(juglone), 6-hydroxy-1,4-naphthoquinone,2,3-dihydroxy-1,4-naphthoquinone, 2,5-dihydroxy-1,4-naphthoquinone,2,6-dihydroxy-1,4-naphthoquinone, 2,7-dihydroxy-1,4-naphthoquinone,2,8-dihydroxy-1,4-naphthoquinone, 5,6-dihydroxy-1,4-naphthoquinone,5,7-dihydroxy-1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone(naphthazarin), 6,7-dihydroxy-1,4-naphthoquinone, and2,3,5,7-tetrahydroxynaphthoquinone (spinochrome B). Other1,4-naphthoqinone derivatives include menadione (sometimes referred toas Vitamin K3 or 2-methyl-1,4-naphthoquinone).

Examples of 9,10 anthraquinolones include the dihydroxy derivatives1,2-Dihydroxyanthraquinone (alizarin), 1,3-Dihydroxyanthraquinone(purpuroxanthin, xantopurpurin), 1,4-Dihydroxyanthraquinone(quinizarin), 1,5-Dihydroxyanthraquinone (anthrarufin),1,6-Dihydroxyanthraquinone, 1,7-Dihydroxyanthraquinone,1,8-Dihydroxyanthraquinone (dantron, chrysazin).2,3-Dihydroxyanthraquinone, 2,6-Dihydroxyanthraquinone, and2,7-Dihydroxyanthraquinone; the trihydroxy-derivatives1,2,3-Trihydroxyanthraquinone (anthragallol),1,2,4-Trihydroxyanthraquinone (purpurin), 1,2,5-Trihydroxyanthraquinone(oxyanthrarufin), 1,2,6-Trihydroxyanthraquinone (flavopurpurin),1,2,7-Trihydroxyanthraquinone (isopurpurin, anthrapurpurin),1,2,8-Trihydroxyanthraquinone (oxychrysazin),1,3,5-Trihydroxyanthraquinone, 1,3,6-Trihydroxyanthraquinone,1,3,7-Trihydroxyanthraquinone, 1,3,8-Trihydroxyanthraquinone,1,4,5-Trihydroxyanthraquinone, 1,4,6-Trihydroxyanthraquinone,1,6,7-Trihydroxyanthraquinone, and 2,3,6-Trihydroxyanthraquinone.

Salt complexes may be formed of FAM and/or AAM compounds as has alreadybeen described. In addition, complexes may also be formed between FAMcompounds and certain AAM compounds. Such FAM+AAM complexes include atleast those with vitamins, such as ascorbates and ascorbylpalmitates;those with amino acids, such as arginates, lysinates, aspartates,glutamates; and those with acidic sugars, such as glucosides, ribosides,galactosides, mannosides, and the like. Example 4 provides a fewconcrete examples of both salts and complexes of FAM and AAM compounds.

The nature of the FAM compounds as liquid crystals may facilitate and/ormediate their role in wound healing. The polar nature of the liquidcrystals allows them to self assemble into polymeric-like structures;they are thus capable of (i) generating their own hydrogels, and/or,(ii) by addition of these molecules to traditional hydrogels, enhancingthe sol-gel transition state to create a unique set of liquid crystalhydrogels. These gels may be modified to create any variety of theaforementioned gel types from strong to weak chemical bonding or thecreation of a biodegradable gel. Without wishing to be bound by anyparticular theory, it is believed that these FAM molecules are useful inwound healing applications, in part because the molecules themselves canbehave like the collagen fibrils that assemble higher order structuresto help with scaffolding and cell migration during wound healing.Further, addition of these liquid crystal hydrogels may facilitateproper collagen alignment and orientation reducing the risk of scar orkeloid formation during the wound healing process.

In addition to hydrogel formulations, another useful formulation of FAMcompounds is with cyclodextrins. A general cylodextrin formula consistsof an FAM with a ratio of FAM: Cyclodextrin of about 1:1, 1.5: 1, 1.5:2,1.5:3, 1:3, 1.5:4, 1:4, 1.5:5, 1:5, 1.5:6, 1:6, 1.5:7, 1:7, 1.5:8, 1:8,1.5:9, 1:9, 1.5:10 or 1:10. These ratios will allow for adequatedissolving of the FAM in cyclodextrin. An AAM may be added from about0.1% to about 99% (w/v), e.g. 0.1-10%, 10-20%, 20-30%, 30-40%, 50-60%,60-70%, and 80-90% (w/v) or higher.

Utility of FAMs and AAMs in Autophagy Modulation

In recent years, scientists have been studying the effects of theregulation of the autophagy pathways as a way to treat a variety ofserious illnesses. In fact, dysregulation of autophagy has been linkedto major diseases including heart disease, cancer and diabetes (See FIG.2, taken from Klionsky D. J. (2010). The Autophagy Connection.Developmental Cell. July 20; 19(1):11-2.) This paper describes the linkbetween the autophagy pathways and major human diseases. While notnaming individual “myopathies,” the ability to regulate this pathway isa major link to modulating disease outcomes. Depending on the diseasestate it may be beneficial to upregulate or downregulate cellular levelsof autophagy.

In some disease states, the pathological origins are related tosuppressed autophagic activity or the activation of autophagy and itsrelated signaling pathways will result in the suppression ofinflammation. Hence, diseases or conditions where it may be beneficialto upregulate autophagy include: wound healing, promote hair regrowth,bacterial infections, inflammation, viral infection, Parkinson'sdisease, Alzheimers, neurodegenerative diseases, neuropathy,cardiovascular disease, heat failure, heart disease, aging, Alzheimer'sdisease, atherosclerosis, arterosclerosis, chronic obstructive pulmonarydisease (COPD), Crohn's disease, inflammatory bowel, colitis, diabetes,diabetes type I and II, amyloidosis, bursitis, dermatitis, angitis,autoimmune diseases with inflammation, blood diseases, aplastic anemia,endometriosis, hepatitis, herpes, HIV, multiple sclerosis, retinaldetachment, age-related macular degeneration, retinitis pigmentosa, andLeber's congenital amaurosis, lysosomal storage diseases, arthritis,psoriasis, osteopenia, osteoporosis, surgical scars, surgical adhesions,space travel (bone density disorder), tendonitis, and ulcerativecolitis.

In other disease states the pathological origins are related tooverexpression of autophagic activity and the activation of autophagyand its related signaling pathways. Hence, diseases or conditions whereit is beneficial to downregulate autophagy include: Aging, Cancer,polycystic kidney and liver disease, kidney disease, liver disease,asthma, diabetic retinopathy, fibromyalgia, ankylosing spondylitis,celiac disease, Grave's disease, lupus, metabolic diseases, nephritis,rheumatoid arthritis, osteolysis, ischemia-reperfusion (I/R) injury,organ and tissue transplant, scleraderma, and sepsis.

In wound healing, increasing autophagy levels assists in tissueprotection, decreases inflammation and promotes the synthesis ofprocollagen, hyaluronan and elastin. As shown herein FAM and AAMcompounds have been used alone and in combination to promote woundhealing, hair growth, and skin repair following damage from UV radiationexposure.

In bacterial infections various types of bacteria attempt to interferewith the autophagy pathway to prevent the cellular uptake of thebacteria ultimately leading to autophagolysosme degradation of thebacteria. Two clinically important skin pathogens, Streptococcus sp. andStaphylococcus aureus interfere with the autophagy pathway (see, I.Nakagawa, et al, Autophagy defends cells against invading group AStreptococcus, Science 306, 1037-1040 (2004); and Schnaith, et al,Staphylococcus aureus subvert autophagy for induction ofcaspase-independent host cell death, J Biol Chem 282, 2695-2706 (2007).Invasive skin infections with group A Streptococcus are characterized bythe prevention of cellular uptake of bacteria due to encapsulation; andif bacteria are taken up by keratinocytes, the majority of streptococciare killed within a few hours (H. M. Schrager, J. G. Rheinwald and M. R.Wessels: Hyaluronic acid capsule and the role of streptococcal entryinto keratinocytes in invasive skin infection, J Clin Invest 98,1954-1958 (1996)). Nakagawa et al. (cited above) showed that autophagyis responsible for the killing activity. Although, some bacteriasurvive, the reduction of the number of extracellular streptococci islikely to have a partially protective effect. As the mechanistic studiesof the action of autophagy against group A Streptococcus have not beenperformed in keratinocytes, additional studies will be necessary tounderstand the relevance and efficiency of this putative antibacterialstrategy in the skin. S. aureus induces autophagy via its alpha-toxin(Schnaith et al, above, and M. B. Mestre, C. M. Fader, C. Sola and M. I.Colombo: Alpha-hemolysin is required for the activation of theautophagic pathway in Staphylococcus aureus-infected cells, Autophagy 6,110-125 (2010)). Pore-forming toxins cause a drop in nutrient and energylevels that trigger autophagy as a rescue mechanism to re-establishcellular homoeostasis (N. Kloft, et al: Pro-autophagic signal inductionby bacterial pore forming toxins, Med Microbiollmmunol 199, 299-309(2010)). Whether autophagy suppresses or enhances S. aureus infection inthe skin in vivo remains to be determined.

EXAMPLES Example 1 Synthesis of Certain FAMs of the Invention:

The following compounds were prepared and given identifying numbers asshown in Table B.

TABLE B Stilbene and Tolan compounds useful in the invention IdentifierCompound Name Structure BM2201 4,4′-dihydroxy-trans-stilbene

BM2301 3,5,4′-trihydroxy-trans-stilbene

BM2401 3,3′,5,5′-tetrahydroxy-trans-stilbene

BM2213 4-hydroxy-4′-(trifluoro)methyl-trans-stilbene

BM3103 4-hydoxy-4′-methoxytolan

BM3302 2,4,4′-trihydroxytolan

BM3032 2,4,4′-trimethoxytolan

BM3203 4,4′-dihydroxy-3-methoxytolan

BM3402 2,4,2′,4′-tetrahydroxy-tolan

BM3206 4,4′-dihydroxytolan-2-O-β-D-glucoside

BM3301 3,5,4′-trihydroxytolan

BM3401 3,3′,5,5′-tetrahydroxytolan

BM3213 4-hydroxy-4′-(trifluoro)methyltolan

Stilbene compounds (BM2xxx series) were synthesized/obtained accordingto the procedures described in Ali, M et al 1992, and Thakkar, K. et al1993, noted above. Tolan compounds (BM3xxx series) were synthesizedaccording to the procedures described in U.S. Pat. No. 6,599,945 B2 ofDocherty & Tsai.

Example 2 Simplified Synthesis Procedures for 4-hydroxy-4′ methoxytolan

Synthesis Procedure for4-Hydroxy-4′-methoxytolan(4-((4′-methoxyphenyl)ethynyl)phenol) was aHeck-type reaction modified from (Pavia, M. R.; Cohen, M. P.; Dilley, G.J.; Dubuc, G. R.; Durgin, T. L.; Forman, F. W.; Hediger, M. E.; Milot,G.; Powers, T. S.; Sucholeiki, I.; Zhou, S.; Hangauer, D. G. The designand synthesis of substituted biphenyl libraries. Bioorg. Med. Chem.1996, 4, 659-666. Jeffery, T. Heck-type reactions in water. TetrahedronLett, 1994, 35, 3051-3054, Jeffery, T.; Galland, J. C.Tetraalkylammonium salts in heck-type reactions using an alkali metalhydrogen carbonate or an alkali metal acetate as the base. TetrahedronLett, 1994, 35, 4103-4106, and Schmidt-Radde, R. H.; Vollhardt, K.;Peter C. The total synthesis of angular [4]- and [5] phenylene J Am ChemSoc, 1992, 114, 9713-9715). The resulting product was a yellowish powderand was verified using ¹HNMR (CDCl₃, 300 MHz): δppm:7.44(d, 4H, J=8.7,Ar-H), 6.89(d, 2H, J=8.7, Ar-H), 6.82(d, 2H, J=8.7, Ar-H), 4.89(s, 1H,OH), 3.85(s, 3H, CH₃O). The resulting product was 98.2% pure and wasused for all subsequent testing.

Example 3 Synthesis Procedure Outline for 2,4,4′-trimethoxytolan

Synthesis was a Heck-type reaction analogous to that of Example 2.

The resulting product was an off white powder and was verified using¹HNMR (CDCl₃, 400 MHz): δppm: 7.47(d, 2H, J=6.4, Ar-H), 7.40(s, 1H,Ar-H), 6.85(d, 2H, J=6.8, Ar-H), 6.47(dd, 2H, J=2.4, Ar-H), 3.89(s, 3H,CH₃O), 3.82(s, 6H, 2CH₃O) and found to be 99.3% pure.

Example 4 Melting Points of FAM Salts and FAM and AAM Salt Complexes

All salts were made by combining sufficient quantities of each stilbene,tolan or combination with magnesium hydroxide, zinc oxide, ascorbic acidor ascorbyl palmitate to generate a salt solution. Each solution wasthen dried in 20 mL scintillation vials using a rotary evaporator(Centrifan, Harvard Biosciences) set to 40° C. and evaporated with amixture of ethanol and ice to allow for a slower evaporation. Onceevaporated and dried completely the salts were ground into a fine powderand their melting points were used to confirm the formation of the salt.Melting points (Table C, below) were determined using a Meltemp IIapparatus outfitted with a temperature probe and thermal couple toprovide a digital read out. Apparatus was calibrated and compounds withknown melting points were tested to confirm calibration prior toanalysis of unknowns.

TABLE C Melting points FAM′s Melting Point 4-hydroxy-4′-methoxytolan140-144° C. 3,5,4′ trihydroxytolan 208-214° C.3,5,3′,5′tetrahydroxystilbene 319-322° C. 2,4,4′ trimethoxytolan  68-73° C. FAM complexes Mg-4-hydroxy-4′-methoxytolan 150-152° C.Mg-3,5,4′ trihydroxytolan 222-232° C. Mg-3,5,3′,5′ tetrahydroxystilbene324-337° C. Mg-2,4,4′ trimethoxytolan   71-76° C. Zn-3,5,4′trihydroxytolan 161-164° C. Zn-3,5,3′,5′tetrahydroxystilbene 217-221° C.Zn-2,4,4′ trimethoxytolan  69-73° C. Zn-4-hydroxy-4′-methoxytolan168-173° C. FAM + AAM complexes Mg-Ascorbate-4-hydroxy-4′- 173-183° C.methoxytolan Zn-Ascorbate-4-hydroxy-4′- 168-175° C. methoxytolan AAM′sAscorbic Acid 190-192° C. Mg(OH)₂    350° C. ZnO   1975° C. AscorbylPalmitate 115-116° C.

Example 5 LC3-II (Microtubule Associated Protein 1 Light Chain 3)Staining in Human Dermal Fibroblasts

Staining method was modified from procedures described by: Furuta, S,(2000) Ras is involved in the negative control of autophagy through theclass I PI3-kinase, Oncogene. 23: 3898-3904; Ge, J. N. et al, (2008)Effect of starvation-induced autophagy on cell cycle of tumor cells,Chinese Journal of Cancer 27:8 102-108; and Settembre, C. et al (2011)TFEB Links autophagy to lysosomal biogenesis, Science 332:17 1429-1433.HDFn (Human dermal fibroblasts neonatal, ThermoFisher) cells were seededat 5,000 cells/well into a black well clear bottom plate (Coning,Corning, N.Y.). Cells were then treated with media alone, FAM, AAM orcombination thereof for 8 hrs. Following treatment time(s) the cellswere fixed with 4% (w/v) PFA (paraformaldehyde) at room temperature for15 min. Cells were then washed with PBS (phosphate buffered saline, pH7.4) and blocked with 5% (w/v) BSA in PBS for 1 hr. Cell were washedagain with PBS and incubated in primary antibody for LC3-IIB (rabbitmonoclonal antibody, Thermo-Fisher) for 3 hrs. Cells were rinsed againin PBS and the secondary fluorescent antibody (Alexafluor 488 rabbitanti goat, ThermoFisher) was added for 30 minutes. The plates were thenimaged using the SpecraMax i3X (Molecular Devices, Sunnyvale, Calif.)and SoftMax Pro 6.5.1 software was used to determine the number ofLC3-II positive cells. Results are shown in FIG. 3

Example 6 Autophagy Modulation and Hormesis

LC3-II Western blot: Human dermal fibroblasts were plated at a densityof 1×10⁶ cells per T25 tissue culture treated flasks. Cells were thentreated with an FAM, AAM or combination for 8 hrs. Cells were thentrypsinized, washed with 1× PBS and lysed with RIPA buffer+proteaseinhibitor on ice. Cells were sonicated and a BCA protein assay (PierceScientific) was run to determine protein concentrations. Concentrationswere then normalized to 100 ug per sample and run on a 12% (w/v)polyacrylamide gel with loading dye and appropriate molecular weightmarkers (All reagents were purchased from National Diagnostics). The gelwas then transferred, blocked and a primary monoclonal antibody toMAPLC3-2 (Thermo-Fisher) was added to the membrane and allowed toincubate overnight. Following appropriate washing a Eu-labeled secondaryantibody (Molecular Devices) was added and the membrane was imaged usingthe Spectramax i3x equipped with the Scan Later module. The gel wassubsequently stripped and re-probed for actin to confirm appropriateloading. SoftMax Pro 6.5.1 software was used to determine changes inaverage band intensity. Results are shown in FIGS. 4 & 5.

Example 7 Autophagy, Wound Healing and the Skin

Very low levels of autophagy are present in the skin, functioning todegrade protein aggregates, damaged organelles and to effect skin colorthrough an FGF-PI3K-AKT-MTOR signaling pathway in the melanosomes(Belleudi, et al. The receptor tyrosine kinase FGFR2b/KGFR controlsearly differentiation of human keratinocytes, PLoS One 2011; 6:e24194;PMID:21957444; http://dx.doi.org/10.1371/journal.pone.0024194; andBelleudi, et al, Expression and signaling of the tyrosine kinaseFGFR2b/KGFR regulates phagocytosis and melanosome uptake in humankeratinocytes. FASEB J 2011; 25:170-81; PMID:20844240;http://dx.doi.org/10.1096/fj.10-162156.) The induction of autophagy inhuman kertinocytes negatively regulates p62, preventing excessiveinflammation and induction of cathelicidin (found in the lysosomes ofmacrophage and PMN's). (Lee, et al Autophagy Negatively RegulatesKeratinocyte Inflammatory Responses via Scaffolding Protein p62/SQSTM1.J Immunol. published online 15 Dec. 2010). In a deep wound second degreeburn model the autophagy inducer rapamycin was shown to enhanceautophagic vesicle formation, improve wound reepithelization times anddecreased IL-8, methane dicarboxylic aldehyde (MDA an indicator ofoxidative stress) and myeloperoxidase (an indicator of the production ofhypochlorous acid (HOCl), hydrogen peroxide (H₂O₂) and chloride anions(Cl—)) levels. (Xiao et al, (2013) Rapamycin reduces burn woundprogression by enhancing autophagy in deep second-degree burn in rats.Wound Rep. Reg. 21: 852-859). This indicates that the induction ofautophagy in the skin creates an anti-inflammatory effect. Rapamycin isa known hormetic chemical that, when used in a dose dependent fashion,can prevent or treat a variety of diseases.

The formulations described in this patent are hormetic substances thathave been shown to induce autophagy in a dose dependent fashion for thetreatment of a variety of diseases and medical conditions, includingincreased wound closure and re-epitheliazation, as shown below.

Example 8 Protein Kinase B (AKT)

HDFn cells were plated in T-25 flasks at a density of 1×10⁶ cells perflasks and allowed to attach and spread overnight. Cells were thentreated for 8 hrs with Control media, FAM, AAM or combination. Cellswere removed using trypsin, followed by trypsin neutralizer and spundown to collect cell pellet. Cells were washed in ice cold 1× PBS andthen lysed with RIPA buffer+protease inhibitor on ice. Samples were spundown, aliquoted and frozen until they could be assayed using an ELISA.Cells were sonicated and diluted 1:5 as recommended in kit instructions.Samples were then assayed using an AKT ELISA (Thermo-Fisher) andconcentrations were determined using an AKT standard curve. Results areshown in FIG. 6.

Example 9 Fibroblast Growth Factor (FGF)

HDFn cells were plated in a 6 well tissue culture treated plate andallowed to reach 70-80% (cells/area) confluency. A cell scraper was thenused to create a “scratch” down the middle of the plate to simulateinjury to the cell monolayer. Dishes were then washed with media toremove any cellular debris. Cells were then treated with Control media,FAM, AAM or combination and samples were then take at 3, 8, and 24 hrs.Samples were spun down, aliquoted and frozen until they could be assayedusing an ELISA. FGF Streptavidin-HRP ELISA kit was used to determineconcentration of EGF in each sample as compared to a known standardcurve. Results are shown in FIG. 7.

Example 10 In Vivo Evaluation of the LCB's for Wound Healing

Twenty, 5-6 week old Balbc/J (stock #000651) male mice were transferredto Jackson Labs in vivo research laboratory in Sacramento, Calif. Themice were ear notched for identification and housed in individually andpositively ventilated polycarbonate cages with HEPA filtered air.Bed-o-cob corn cob bedding was used and cages were changed every twoweeks. The animal room is lighted entirely with artificial fluorescentlighting, with a controlled 12 h light/dark cycle. The normaltemperature and relative humidity ranges in the animal rooms are 22±4°C. and 50±15%, respectively. The animal rooms were set to have 15 airexchanges per hour. Filtered tap water, acidified to a pH of 2.8 to 3.1,and rodent chow was provided ad libitum. Following a 5-7 daysacclimation, mice were randomized by body weight into 2 cohorts of 10mice each. On study day 0, mice were anesthetized and two full thicknessexcision wounds (˜6 mm) were made on the dorsum (backs) of mice. One ofthe wounds was covered using a semi-occlusive polyurethane dressing(Tegaderm™). Dressings covered the wounds for 5 consecutive (5) daysfrom the day of wounding (d 0). The doses of 4-hydroxy-4′-methoxytolanwere based on those that promoted lesion healing in mice in previousherpes virus studies. Wound measurements were made on day 5, 7, 9 and11. Digital images of wounds were taken of each mouse. Test agents werea viscous paste and wounds were surrounded with a saddle glued in placeto eliminate cross contamination and wound closure due to contraction.Therefore, changes in wound area are due to re-epithelization. Duringthe five days all wounds are covered with glycerin (control) or glycerinand test agent. At day 5, test agents were removed and changes in woundarea were measured (as area/area %) in an effort to gauge thepersistence of the compounds in the wound site. By day 5, glycerinproduced 26.1% wound closure, whereas 4-hydroxy-4′-methoxytolan (2.5%)closed produced 92.8% wound closure and 5% 4-hydroxy-4′-methoxytolanproduced 91.4% wound closure. Prior to statistical analysis a test forNormality was run and a Z-score computed to confirm a normallydistributed area for each wound on day 1 for all mice across groups,r²=0.989. Statistics are computed based One-Way ANOVA's comparingchanges in wound area on day 5. All mice tolerated the treatments andall weekly clinical observations report bright, alert, responsive, andhydrated mice.

TABLE D Wound size reduction Day Average Area (cm²) Wound Area (%)ClosedControl Animals 0 2.11 0 5 1.56 26.1 7 0.63 70.2 9 0.55 73.9 11 0.4976.8 4-hydroxy-4′- 0 1.98 0 methoxytolan 5 0.17 91.4 5% (w/v) 7 0.08 1009 0.00 100.0 11 0.00 100.0 Day Average Area (cm²) Wound Area (%)4-hydroxy-4′- 0 2.07 0 methoxytolan 5 0.15 92.8 2.5 % (w/v) 7 0.04 100 90.00 100.0 11 0.00 100.0

Example 11 Excised Skin Samples Treated with a Control or with 5% (w/v)4-hydroxy-4′-methoxytolan

H&E staining of control treated cells revealed a keratinocyte layer of1-2 cells thick on top of a loosely organized layer of myofibroblasts,connective tissue with a few deep sebaceous glands beginning to form.4-hydroxy-4′-methoxytolan treated skin revealed-extensive keratinocyteproliferation with cell layers 7-8 cells thick. The dermis showedregular and proliferative myofibroblasts and connective tissuesurrounding highly proliferative and prevalent sebaceous glandsextending to the surface to reestablish hair follicles.

Example 12 Autophagy and Hair Re-Growth in Skin Appendages (Hair Loss)

Autophagosome-like structures have been detected by electron microscopyof hair and sebaceous glands. While the physiological relevance ofautophagy in skin appendages is not well understood at present, currentexisting data suggests induction of autophagy may prevent hairlossthrough a Wnt1 dependent cell rejuvenating process where damaged cellsundergo cell death and hair stem cell are stimulated to generate hairgrowth (Castilho R. M., et al. (2009) mTOR mediates Wnt-inducedepidermal stem cell exhaustion and aging, Cell Stem Cell 5, 279-289; andVishnyakova, et al. (2013) Possible Role of Autophagy Activation inStimulation of Regeneration, Molecular Biology. 47(5): 692-700).

Example 13 UV Radiation Damage and Anti-Aging

The skin is the largest organ in the human body and is in contact withthe environment. As such it is constantly subjected to damage, both fromoutside and from the inside, which threatens its balance and alters itsappearance. This damage is often manifested as chronic low levels ofinflammation. It is known for example that excessive exposure to UV isreflected by various cutaneous manifestations, such as actinicerythemas, solar elastosis, or else the premature appearance of theeffects of cutaneous aging: the skin becomes loose, deeply wrinkled,rough, dry, sprinkled with hypopigmented or hyperpigmented spots anddilated vessels. These manifestations of UV exposure, which reflectprofound structural changes in the cutaneous tissue, are unsightly andugly, and many people have a tendency to want to smooth them out. TEMand IF microscopy analysis of cultured dermal fibroblasts from women ofdifferent ages revealed an impaired autophagic flux. When young dermalfibroblasts were treated with lysosomal protease inhibitors to mimic thecondition of aged dermal fibroblasts the reduced autophagic activity,altered the fibroblast content of type I procollagen, hyaluronan andelastin, and caused a breakdown of collagen fibrils. Together thesefindings suggest that impaired autophagic induction leads todeterioration of dermal integrity and skin fragility (Tashiro K, et al.(2014) Age-related disruption of autophagy in dermal fibroblastsmodulates extracellular matrix components, Biochem Biophys Res Commun.443(1): 167-172).

Autophagy ensures that damaged cellular organelles and proteinaggregates are degraded properly and do not accumulate causing cellulardysfunction. Enhanced autophagic activity is also seen in response tocaloric restriction (CR) which has shown to prolong life expectancy.

Example 14 A 40% (w/v) Solution of Hydroxypropyl-β-cyclodextrin

Solution is prepared by adding into a sterile graduated beaker 40.0 g ofHydroxypropyl-β-cyclodextrin to 70 mL of water and mixed thoroughly.Once the solution is clear QS to 100 mL. Weigh out 1.0 g of the liquidcrystal compound (FAM) and transfer into a sterile glass bottle. Add 1.5mL of ethanol to the bottle and dissolve completely. Slowly add 1 mL ofcyclodextrin while stirring to ensure drug remains in solution. Add 5 mLof water while stirring to ensure drug stays in solution. Sonicate ifnecessary. Formulation should be a clear solution. Filter using a 0.2 umfilter. The suspension is frozen below −40° C. and is lyophilized. Thelyophilized cake maybe reconstituted with sterile water prior to use.

Example 15 Preparation of an Injectable Liquid Crystal Formulation FAMCyclodetrin Formulation

100 mg of a 4,4′-dihydroxytolan compound are weighed and placed in a 5ml scintillation tube. 1.5 ml of absolute ethanol is added to the tubeand shaken until the 4,4′-dihydroxytolan is completely dissolved. 5grams of pyrogen free hydroxypropyl-β-cyclodextrin (Sigma) are weighedon an analytical scale and placed in a graduated cylinder. Water isadded with shaking until the volume reaches 90 ml. The above ethanolicsolution of FAM is added to the aqueous solution containinghydroxypropyl-β-cyclodextrin with stirring. Water is added to the clearsolution to make the total volume 100 ml. The solution is sterilefiltered through a 0.22 micron filter. The suspension is frozen below−40° C. and is lyophilized. The lyophilized cake is reconstituted withsterile water for injection prior to use.

Example 16 Preparation of Drop Solution

The solution is compounded from the ingredients; FAM-cyclodextrin 0.625parts; saccharin sodium 0.3 parts; sorbic acid 0.1 parts; ethanol 30.0parts; flavoring 1.0 parts; distilled water q.s. ad 100.0 parts. TheFAM-cyclodextrin complex and the flavoring are dissolved in the ethanol,and the sorbic acid and the saccharin are dissolved in the distilledwater. The two solutions are uniformly admixed with each other, and themixed solution is filtered until free from suspended matter: 1 ml of thefiltrate contains the FAM and is an oral dosage unit composition witheffective therapeutic action.

Example 17 Preparation of Micronized Drug and Drug Suspensions

16 grams of micronized FAM is milled with a 4 inch mill size andcompressed nitrogen gas/compressed air (dew point >40° C.) as millinggas. The material is manually fed into the hopper and placed on top ofthe feed tray. The material is drawn into a confined, circular chamberby way of a pressurized milling gas. The powder becomes suspended in ahigh velocity stream in the milling chamber. Particle size distributionis measured on a particle size analyzer. The milling conditions are thenadjusted to give material with an acceptable micron size.

Example 18 An FAM or AAM Modulator-Cyclodextrin Complex Gel

100 mg of an FAM is weighed and placed in a sterile test tube. The FAMis dissolved in 2-3 ml of purified absolute ethanol. 50 ml of a 10-50%(w/v) solution of hydroxypropyl-β-cyclodextrin (other cyclodextrans mayalso be used based on the need for water absorption such asα-cyclodextrins, γ-cyclodextrins and certain modified β-cyclodextrins)is prepared in a 150 ml sterile beaker and the solution is heated to70-80° C. while stirring on a hot plate. The ethanolic solution of FAMis slowly added to the beaker with stirring. At this stage the AAM maybe added from 1-25% (w/v). The addition of the FAM will start thegel-sol transition and if desired a gelling molecule such as sodiumpectate dissolved in deionized water can be added to further enhancegelation. Other gel enhancers include the monovalent or divalent cationFAM or AAM salts which will form ionic cross linkages to enhance gelformation.

The use of cations can be selected based on the desire to increase ordecrease solubility in water. In order to enhance the gelation processan FAM or its magnesium dimer is added to alginate copolymers duringmixing and the M/G ratio is adjusted to create stabile FAM:Mg²⁺:Alginatebiodegradable sheets. The FAM cation interacts through hydrogen bondingwith the pocket created by the G form alginate copolymers (see FIG. 15).All ratios can be adjusted to optimize FAM concentration andpolymerization. When necessary in addition to Mg²⁺ other ions such asCa²⁺, K⁺ or Zn²⁺ may be added further enhance the gelling process.

Alginates G, M or G/M copolymers through the addition of divalentcations such as calcium form calcium alginate sheets. These sheets canbe created in a sterile environment and are non-irritating,non-sensitizing and biodegradable. This makes liquid crystal FAMmolecules ideal molecules to promote alginate gelation, polymerization,control copolymer block structure and alter acetylation to influence thephysicochemical and rheological characteristics of the polymer. Inaddition to adjusting the molecular mass of the individual alginatemonomers the selected FAM's may also alter gel viscosity.

A variety of polymeric sugar molecules that when combined with liquidcrystal FAM and AAM molecules described in this application can createunique hydrogels, alginates and drug delivery systems that can be usedin creating novel wound care products. Examples include but are notlimited to chitosan, hyaluronic acid, pectin, heparin, alginate,chondroitin sulfate A, D &E, PEG (polyethylene glycol), PLA (polylacticacid) and polymers thereof and polyphosphazene.

In certain instances an additional FAM or AAM may be added to any of theabove formulations to improve solubility, adjust pH, balance cation oranion concentration, improve adherence to the skin, increase or decreasesolubility in water, create a concentration gradient, improve gel-soltransition, increase or decrease electrical conductivity, increase ordecrease capacitance, adjust overall resistance or impedance.

The formulations described in this patent are liquid crystal hormeticsubstances that have been shown to induce autophagy in a dose dependentfashion for the treatment of a variety of diseases and medicalconditions, including increased wound closure and re-epitheliazation.

Example 19 An FAM or AAM Modulator-Alginate Complex

One gram of sodium alginate is dissolved in deionized water in a 500 mLbeaker and to that is added if desired a selected AAM (1-25% w/v) isadded while mixing. To this solution is added a therapeuticallyeffective amount of an FAM that was previously dissolved in ethanol to adesired concentration 1-50% (w/v) and if necessary the addition of amonovalent or divalent cationic salt is added until the gel has reacheddesired consistency. For salts of FAM's the previous step is notrequired. The liquid crystal nature of the FAM molecules creates uniqueco-block polymers that associate through hydrogen, electrostatic andionic bonding. This solution can then be used to coat woven cotton orother fibers to create alginate bandages. This same solution can also beused to create biodegradable sheets, films, beads or gels.

The foregoing description of the various aspects and embodiments of thepresent invention has been presented for purposes of illustration anddescription. It is not intended to be exhaustive of all embodiments orto limit the invention to the specific aspects disclosed. Obviousmodifications or variations are possible in light of the above teachingsand such modifications and variations may well fall within the scope ofthe invention as determined by the appended claims when interpreted inaccordance with the breadth to which they are fairly, legally andequitably entitled.

What is claimed is:
 1. A method for promoting wound healing in a patienthaving a wound or skin condition, comprising administering to a patienta therapeutically effective amount of a formulation comprising: a firstautophagy modulating compound having the structure (I):

wherein L is a linker comprising —C≡C—; R¹ and R² are independentlysubstituents at any available position of the phenyl rings; m and n are,independently, 0, 1, 2, or 3 representing the number of substituents onthe rings, respectively, and at least one of m or n must be ≥1; whereineach R¹ and R² is independently selected from: —R⁵, wherein R⁵ isselected from (C₁-C₆)alkyl, (C₂-C₆)alkenyl, or (C₂-C₆)alkynyl;optionally substituted with 1 to 3 substituents selected from —OH, —SH,-halo, —NH₂, or NO₂; —YR⁶, wherein Y is O, S, or NH; and R⁶ is selectedfrom H or R⁵; —ZR⁵, wherein Z is —N(C═O)— or —O(C═O)—; -halo; —NO₂;—SO₃Na; -azide; and -glycosides -and salts thereof; with the provisothat the first autophagy modulating compound is not resveratrol or4,4′-(ethyne-1,2-diyl)diphenol (TOLECINE, also known as4,4′-dihydroxytolan).
 2. The method of claim 1, wherein the firstautophagy modulating compound is a hydroxylatled tolan, having from 1 to4 hydroxyl substituents.
 3. The method of claim 1, wherein the firstautophagy modulating compound is selected from: 2,4,4′-trihydroxytolan;4,3′,5′-trihydroxytolan; 2,2′,4,4′-tetrahydroxytolan;3,3′,5,5′-tetrahydroxytolan; 4-hydroxy-4′-(trifluoro)methyltolan;4,4′-dihydroxy-3-methoxytolan; 2,4,4′-trihydroxytolan-beta-D-glucoside;and 4-hydroxy-4′-methoxytolan.
 4. The method of claim 1, furthercomprising co-administering an auxiliary autophagy modulating compound.5. The method of claim 4, wherein the auxiliary autophagy modulatorcompound is administered at the same time as the first autophagymodulator compound.
 6. The method of claim 4, wherein the auxiliaryautophagy modulator compound is administered prior to administering thefirst autophagy modulator compound.
 7. The method of claim 4, whereinthe auxiliary autophagy modulating compound is selected from the groupconsisting of: substituted or unsubstituted parabenzoquinone,substituted or unsubstituted orthobenzoquinone, substituted orunsubstituted anthraquinone, an amino acid, an acidic monosaccharide,and a vitamin or a salt thereof.
 8. The method of claim 7, wherein thevitamin is an oxygen-containing vitamin or an isoprenoid-containingvitamin.
 9. The method of claim 1, wherein the first autophagymodulating compound upregulates autophagy activity.
 10. The method ofclaim 1, wherein the wound or skin condition is one or more selectedfrom aging, autoimmune diseases with inflammation, avascular necrosis,bacterial infection, cancers, diabetic neuropathies, endometriosis,fungal infection, gout, hairloss, infectious arthritis, inflammation,inflammatory bowel, ischemia, Lyme disease, organ/tissue transplant,parasitic infection, psoriatic arthritis, psoriasis, pseudogout,rheumatoid arthritis, scleraderma, scurvy, sepsis, skin diseases,surgical scars, surgical adhesions, transfection procedures, ulcerativecolitis, ulcers, viral infection, warts, surgical wounds, incisions,lacerations, cuts and scrapes, donor site wounds from skin transplants,traumatic wounds, infectious wounds, ischemic wounds, burns, bullouswounds, aseptic wounds, contused wounds, incised wounds, laceratedwounds, non-penetrating wounds, open wounds, penetrating wounds,perforating wounds, puncture wounds, septic wounds, subcutaneous wounds,chronic ulcers, gastric ulcers, skin ulcers, peptic ulcer, duodenalulcer, gastric ulcer, gouty ulcer, hypertensive ischemic ulcer, stasisulcer, sublingual ulcer, submucous ulcer, symptomatic ulcer, trophiculcer, tropical ulcer, veneral ulcer, hyperkeratosis, photo-aging,psoriasis, skin rashes, sunburns, photoreactive processes, mouth soresand burns, post-extraction wounds, endodontic wounds, ulcers and lesionsof bacterial or viral or autoimmunological origin, mechanical wounds,chemical wounds, thermal wounds, infectious and lichenoid wounds, herpesulcers, stomatitis, aphthosa, acute necrotizing ulcerative gingivitis,burning mouth syndrome, corneal ulcers, radial keratotomy, cornealtransplants, epikeratophakia, surgically induced wounds in the eye,hemorrhoids, pruritus, proctitis, anal fissures, dry cracked skin,seborrheic conditions, anthrax, tetanus, gas gangrene, scalatina,erysipelas, sycosis barbae, folliculitis, impetigo contagiosa, andimpetigo bullosa.
 11. The method of claim 1, wherein the first autophagymodulating compound is dispersed in a hydrogel.
 12. The method of claim11, wherein the hydrogel is a liquid crystalline hydrogel formed in partby the first autophagy modulating compound.
 13. The method of claim 11,wherein the hydrogel is an alginate.
 14. The method of claim 11, whereinthe formulation comprises the first autophagy modulating compoundcombined with a cyclodextrin in a ratio of first autophagy modulatingcompound:cyclodextrin from about 1:1 to about 1:10.
 15. The method ofclaim 11, wherein the formulation comprises the first autophagymodulating compound combined in a complex with ascorbate and a cation.16. A method for promoting wound healing in a patient having a wound orskin condition, comprising administering to a patient a therapeuticallyeffective amount of a formulation comprising: a first autophagymodulating compound having the structure (I):

wherein L is a linker comprising —CR_(a)═CR_(b)—; R_(a) and R_(b) areindependently H or phenyl optionally substituted with —(R³)_(p) or'(R⁴)_(q); R¹ to R⁴ are independently substituents at any availableposition of the phenyl rings; m, n, p and q are, independently, 0, 1, 2,or 3 representing the number of substituents on the rings, respectively,and at least one of m or n must be ≥1; wherein each R¹, R², R³, and R⁴is independently selected from: —R⁵, wherein R⁵ is selected from(C₁-C₆)alkyl, (C₂-C₆)alkenyl, or (C₂-C₆)alkynyl; optionally substitutedwith 1 to 3 substituents selected from —OH, —SH, -halo, —NH₂, or NO₂;—YR⁶, wherein Y is O, S, or NH; and R⁶ is selected from H or R⁵; —ZR⁵,wherein Z is —N(C═O)— or —O(C═O)—; -halo; —NO₂; —SO₃Na; -azide; and-glycosides -and salts thereof; with the proviso that the firstautophagy modulating compound is not resveratrol or4,4′-(ethyne-1,2-diyl)diphenol (TOLECINE, also known as4,4′-dihydroxytolan).
 17. The method of claim 16, wherein the firstautophagy modulating compound is a trans-stilbene wherein L is —CH═CH—.18. The method of claim 17, wherein the stilbene is a hydroxylatedstilbene, having from 1 to 4 hydroxyl substituents.
 19. A method formodulating autophagy in a patient in need of autophagy modulation,comprising administering to a patient a therapeutically effective amountof a formulation comprising: a first autophagy modulating compoundhaving the structure (I):

wherein L is a linker selected from: —C≡C— and —CR_(a)═CR_(b)—; R_(a)and R_(b) are independently H or phenyl optionally substituted with—(R³)_(p) or —(R⁴)_(q); R¹ to R⁴ are independently substituents at anyavailable position of the phenyl rings; m, n, p and q are,independently, 0, 1, 2, or 3 representing the number of substituents onthe rings, respectively, and at least one of m or n must be ≥1; whereineach R¹, R², R³, and R⁴ is independently selected from: —R⁵, wherein R⁵is selected from (C₁-C₆)alkyl, (C₂-C₆)alkenyl, or (C₂-C₆)alkynyl;optionally substituted with 1 to 3 substituents selected from —OH, —SH,-halo, —NH₂, or NO₂; —YR⁶, wherein Y is O, S, or NH; and R⁶ is selectedfrom H or R⁵; —ZR⁵, wherein Z is —N(C═O)— or —O(C═O)—; -halo; —NO₂;—SO₃Na; -azide; and -glycosides; and salts thereof.
 20. The method ofclaim 19, wherein the patient suffers from a condition in need ofautophagy upregulation, said condition comprising one or more of: woundhealing, hair regrowth, bacterial infections, inflammation, viralinfection, Parkinson's disease, neurodegenerative diseases, neuropathy,cardiovascular disease, heat failure, heart disease, aging, Alzheimer'sdisease, atherosclerosis, arterosclerosis, chronic obstructive pulmonarydisease (COPD), Crohn's disease, inflammatory bowel, colitis, diabetes,diabetes type I or II, amyloidosis, bursitis, dermatitis, angitis,autoimmune diseases with inflammation, blood diseases, aplastic anemia,endometriosis, hepatitis, herpes, HIV, multiple sclerosis, retinaldetachment, age-related macular degeneration, retinitis pigmentosa,Leber's congenital amaurosis, lysosomal storage diseases, arthritis,psoriasis, osteopenia, osteoporosis, surgical scars, surgical adhesions,space travel (bone density disorder), tendonitis, ulcerative colitis,aging, cancer, polycystic kidney and liver disease, kidney disease,liver disease, asthma, diabetic retinopathy, fibromyalgia, ankylosingspondylitis, celiac disease, Grave's disease, lupus, metabolic diseases,nephritis, rheumatoid arthritis, osteolysis, ischemia-reperfusion (I/R)injury, organ and tissue transplant, scleraderma, and sepsis.